Method of making hepatocyte-selective oil-in-water emulsion

ABSTRACT

A heat and shelf-stable oil-in-water emulsion useful as a tissue or cell-selective delivery vehicle. Radioactive or stable, synthetic or semi-synthetic polyhalogenated triglycerides, such as 2-oleoylglycerol-1,3-bis[7-(3-amino-2,4,6-triiodophenyl)heptanoate] or 2-oleoylglycerol-1,3-bis[ω-(3,5-bis-trifluoromethyl)hepatanoate] or phenyl acetate, can be incorporated into the lipophilic core of a lipoprotein-like emulsion particle. The lipophilic core is surrounded by a phospholipid membrane comprising cholesterol and apolipoproteins. For hepatocyte-selective delivery, the emulsion is chylomicron remnant-like by being in a size range of 50 to 200 nm as measured by number weighting analysis with a narrow size distribution (&lt;2% greater than 300 nm) and having a composition simulating naturally-occurring chylomicron remnants. Use of cholesterol in the emulsion formula facilitates association of apolipoproteins, especially Apo E which are recognized by liver cells and necessary for binding and uptake. Stability and size constraints can be achieved by using ultra high energy mixing equipment, such as such as the MicroFluidizer (Microfluidics Corp., Newton, Mass.) to form the emulsion. In preferred embodiments, osmolality of the emulsion is controlled by glycerol rather than normal saline.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Grant No. CA08349by the National Institutes of Health. The government has certain rightsin the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/243,596 filed onMay 16, 1994, now U.S. Pat. No. 5,851,510 issued on Dec. 22, 1998.

BACKGROUND OF THE INVENTION

This invention relates generally to an oil-in-water emulsion, and moreparticularly, to an oil-in-water emulsion which functions as atissue-specific delivery vehicle for lipophilic or amphipathicdiagnostic, therapeutic, or other bioactive or inactive agentsincorporated therein.

Imaging agents are used for diagnostic modalities, such as computedtomography (CT), magnetic resonance (MR), ultrasound or nuclearmedicine, to enhance the image contrast between tissue types. It is ashortcoming in the present state of the art that most of the currentlyused imaging agents are limited in action to the vascular and/orextracellular compartments. Thus, every tissue that receives a normalblood supply will also receive the diagnostic agent. Tissue-specificimage enhancement is therefore compromised. Non-specific agents whichreside in the extracellular space are useful primarily to discriminateanatomical features of tissues and structures. However, an imagingagent, which can deliver a diagnostic agent to the intracellularenvironment of a targeted tissue, could provide a means of assessing themetabolic and/or biochemical activity of the targeted tissue in additionto providing the standard anatomical visualization achieved withextracellular imaging agents.

In addition to the foregoing, agents which localize in the extracellularspaces are cleared very rapidly from the body. Due to imaging hardwarelimitations, a predetermined minimum period of time is required tocollect the data that are used to form a diagnostic image. Consequently,a contrast agent that clears too quickly from the body must beadministered in a very large dose in order to maintain a concentrationgradient sufficient to achieve an acceptable quality of the image. Thus,the administration of many currently available diagnostic imaging agentsis a complicated process balancing the benefits of image enhancementagainst the dangers of injecting large volumes of material into a livingbeing in a short period of time. In the case of CT imaging, diagnosticimaging agents are commonly administered to the patient in volumes aslarge as 150 to 250 ml at rates of 1.5 to 2.5 ml/sec. Injection ofcurrently available agents at this rate can induce nausea, headaches,convulsions and other undesirable and dangerous side effects. There isthus a need for a tissue-specific delivery vehicle that concentrates theimaging agent in a single targeted organ or tissue type and thus permitsslower, controlled injection of a substantially smaller dose. Of course,a lower dose would also minimize the potential for toxicity and sideeffects, as well as preclude the need for expensive power injectors.

For therapeutic purposes, such as the delivery of radioactivetherapeutic agents, it would be advantageous to target specific tissueand reduce the destructive effects of the radioactive agents onsurrounding tissue. There has been much discussion of gene therapy fortreatment of such diseases as familial hypercholesterolemia, hepatitis,or hepatomas. However, the delivery of corrective genetic material toabnormal tissues frequently fails because of an inability to target thegenetic material to a specific tissue or to do so in sufficientquantities to replace the abnormal form of the gene. There is,therefore, a need in the art for a delivery vehicle which is capable ofdelivering genetic material to tissues at levels suitable for genetherapy.

Some known strategies for achieving tissue-specific delivery include theuse of vehicles such as liposomes, antibody-linked conjugates andcarbohydrate derivatives of the targeting compound. However, many ofthese known vehicles cannot form acceptable complexes with the moiety tobe delivered or fail to accumulate the complexed moiety in the targettissue in quantities sufficient to be effective for imaging and/ortherapy.

One of the most accurate, non-invasive radiologic examination techniquesavailable for detection of hepatic masses is CT using water soluble,urographic contrast agents. However, the contrast agents commonly usedin this well-known technique suffer from the typical limitations whichplague other known contrast agents, including, for example, therequirement that large doses be administered, a nonspecificbiodistribution, and an extremely short (<2 min.) residence time in theliver. As a result, CT has not been consistently successful at detectinglesions smaller than about 2 cm in diameter. These significantlimitations of the known agents preclude early detection and therapy ofcancer, since many metastases are smaller than the detection limits ofthe technique.

The inability of water-soluble urographic agents to detect lesions lessthan 2 cm in diameter with acceptable consistency may be due, in part,to the rapid diffusion of these agents out of the vasculature intointerstitial spaces resulting in a rapid loss of contrast differentialbetween normal liver tissue and tumors. There is, therefore, a need fora diagnostic contrast agent, or vehicle therefor, which delivers agentto the intracellular space of specific targeted tissue, such as livertissue, so as to enhance the degree of selective visualization andfurther improve the detection limits of CT. A number of alternatives towater-soluble contrast agents have been investigated as potential liverCT contrast agents including, for example, radiopaque liposomes,iodinated starch particles, perfluoroctylbromide, iodipamide ethylesters, and ethiodized oil emulsion (EOE-13). All of these agents areparticulate in nature and of such a size for which liver specificity ismediated primarily via sequestration by the reticuloendothelial system(RES).

Liposomes, which are artificially prepared lipid vesicles formed bysingle or multiple lipid bilayers enclosing aqueous compartments areparticulate in nature, and hence, have potential for delivering agentscontained therein to the RES. Investigators have attempted to loadliposomes with both ionic and non-ionic water-soluble urographic orhepatobiliary contrast agents, or to incorporate brominatedphosphatidylcholine into the bilayer membrane. However, stabilization ofthe resulting liposome against loss of contrast media from the bilayershas proven to be a major problem. Moreover, incorporation of neutrallipophilic agents into the bilayer is limited by the low solubility ofthe lipophilic agents in the membrane matrix and the restricted loadingcapacity of the liposoine.

Several monobrominated perfluorocarbons have been evaluated as contrastagents in animals. The most common of these, perfluoroctylbromide, hasbeen shown to concentrate in the reticuloendothelial cells of the liver,spleen and other organs. The long residency times (weeks to months) andthe large doses (5-10 g/kg) necessary for suitable opacification willmost likely preclude the use of monobrominated perfluorocarbons inhumans for diagnostic imaging purposes unless a means of specificallydelivering small doses to a targeted organ is developed.

The most promising of the investigational agents mentioned above,EOE-13, has been extensively studied in both animals and humans in theUnited States. EOE-13, an emulsion of iodinated poppy seed oil (37%iodine by weight) in saline, offered considerable improvement in thedetection of space-occupying lesions in the liver and spleen as comparedto conventional water-soluble urographic agents. Despite acceptableclinical diagnostic efficacy, a high incidence of adverse reactions,including fever, chills, thrombocytopenia, hypotension, and respiratorydistress, was reported. Moreover, additional problems were encounteredin the sterilization of the EOE-13 preparation. These problems led tothe discontinuation of the use of EOE-13 in humans.

Recently, investigators have demonstrated a direct relationship betweenemulsion particle size and macrophage involvement. The investigatorstested three iodinated lipid emulsions, including EOE-13, having meanparticle diameters ranging from 400-2000 nm. They observed a markedswelling of Kupffer cells which, when coupled with sinusoidalendothelial damage, resulted in sinusoidal congestion. Sinusoidalcongestion often activates macrophages, resulting in the release oftoxic mediators which may be responsible, in part, for the adversereactions seen with these relatively large-sized particulatepreparations. As a result, the investigators emulsified iodinated ethylesters of fatty acids derived from poppyseed oil (Lipiodol-UF,Laboratoire Guerbet, France) with egg yolk phospholipids, in order toprovide a preparation of smaller, more uniform particle size, calledIntraiodol (not commercially available; see, for example, ActaRadiologica, Vol. 30, pages 407-412 and 449-457, 1989). Intraiodol has aparticle size range of 110 to 6550 nm (distribution mean diameter 310nm). Initial results obtained with Intraiodol in animals and humansdemonstrated a significant reduction in adverse reactions relative tothose observed with EOE-13. However, Intraiodol continues to suffer frommany disadvantages common in the prior art, including failure to achievetrue specificity due to, inter alia, liposome (particulate)contamination which results in delivery to the RES, size andcomposition, and inability to achieve shelf and heat stability.Moreover, the iodine necessary for CT opacification is attached in analiphatic linkage, which is well known to exhibit diminished in vivostability.

Although Intraiodol, and other similar oil-in-water emulsions, have beencalled "chylomicron remnant-like" and "hepatocyte specific," theseagents locate significantly in the spleen, which does not containhepatocytes. A true hepatocyte-specific contrast agent will not locatesubstantially in the spleen and other RES associated organs unless thereis saturation of the initial receptor-mediated process so that there isa shift in delivery to the cells of the RES. Further, a truehepatocyte-specific agent will be cleared primarily through the biliarysystem. None of the aforementioned emulsions demonstratehepatocyte-specificity with biliary clearance studies.

Accordingly, there remains a great need in the art for target-specificdelivery vehicles or compositions, including contrast-producingoil-in-water emulsions, for delivery of diagnostic, therapeutic, andother biologically active or inactive agents.

It is, therefore, an object of this invention to provide an improveddelivery vehicle, specifically a target-selective oil-in-water emulsionfor delivery of lipophilic agents, or lipophilic derivatives of watersoluble agents, such as contrast agents, to the intracellular spaces ofthe targeted tissue.

It is another object of this invention to provide a target-specificdelivery vehicle, specifically a hepatocyte-selective oil-in-wateremulsion, which is chylomicron remnant-like with respect to size andbiodistribution characteristics.

It is also an object of this invention to provide a target-selectiveoil-in-water emulsion which is shelf stable and heat stable so that itcan be heat sterilized.

It is a further object of this invention to provide a method ofpreparing a target-selective oil-in-water emulsion which is chylomicronremnant-like, shelf and heat stable, and substantially free of liposomecontamination.

SUMMARY OF THE INVENTION

The foregoing and other objects are achieved by this invention which isa synthetic oil-in-water lipid emulsion resembling endogenouslipoproteins in order to take advantage of the natural lipid transportsystem of a living being. The oil-in-water emulsion of the presentinvention is useful as a target-selective delivery vehicle. In specificembodiments, the oil-in-water emulsion simulates chylomicron remnants sothat lipophilic or amphipathic compounds inserted into either the lipidcore or the surrounding monolayer of the emulsion are deliveredselectively to the hepatocytes via a receptor-mediated pathway. As usedherein the term "receptor-mediated" means that metabolism of theoil-in-water emulsions of the present invention mimics the uptake andclearance of natural chylomicron remnants.

To summarize briefly the natural lipid transport system, lipids aretransported in the plasma mainly in the form of free fatty acids,triglycerides, and cholesteryl esters. Free fatty acids are transportedas a complex with plasma albumin, whereas triglycerides and cholesterylesters are transported in the lipophilic core of plasma lipoproteins.The surface of plasma lipoproteins comprises a monolayer ofphospholipid, cholesterol, and specific proteins known asapolipoproteins which regulate the entry and exit of particular lipidsat specific targets. Cholesterol and triglycerides from dietary sourcesare absorbed by the intestinal tract and incorporated into chylomicronswhich are subsequently secreted into and transported through thethoracic duct until they reach the circulation. Once in circulation,there is a rapid transfer of apoprotein C-II from circulating highdensity lipoproteins to the chylomicrons. Once associated withapoprotein C-II, the chylomicrons are acted upon by lipoprotein lipasein the capillary beds of several peripheral tissues including adipose,muscle (skeletal and heart), and lung. Lipoprotein lipase hydrolyzesmuch of the core triglyceride to glycerol and free fatty acids, most ofwhich are taken up by the tissues for storage or oxidation. Theremaining triglyceride-depleted chylomicrons, called chylomicronremnants, now contain lesser amounts of triglycerides, with cholesterylesters as the main lipid component and apoprotein B (Apo B) andapoprotein E (Apo E) as the major apoprotein components. Chylomicronremnants are cleared very rapidly from the circulation by the liver viaa receptor-mediated process that recognizes Apo B and Apo E. Whilelipoprotein lipase is the enzyme responsible for the hydrolysis ofplasma triglycerides in extrahepatic tissues, hepatic triglyceridelipase is implicated in hepatic triglyceride hydrolysis and remnantuptake by hepatocytes. Hepatic clearance of chylomicron remnants fromthe circulation occurs mainly via the hepatocytes (parenchymal cells)rather than Kupffer cells (nonparenchymal cells).

Therefore, the synthetic oil-in-water emulsion of the present inventionis designed to associate with plasma apoproteins such as Apo B and Apo Eso as to be "hepatocyte-selective." The oil-in-water emulsion of thepresent invention is termed "hepatocyte-selective" in comparison to"hepatocyte-specific" emulsions as that term is used in the prior art,without definition.

In order to achieve Apo I, association, however, the oil phase particlesmust have the correct size and composition, and must retain the correctsize after sterilization and/or over the period of its shelf life. Inaccordance with the present invention, the mean oil phase particle sizeis between 50 and 200 nm (number weighted), with a narrow sizedistribution (50 to 300 nm) wherein no more than 2% of the particleshave a diameter that falls outside of the range (i.e., being greaterthan 300 nm). The emulsion should have no detectable particles with adiameter greater than 1 μm. Moreover, the emulsion should not becontaminated with liposomes.

In addition, the composition must contain a sterol, which is preferablycholesterol, in an amount of up to 5% by weight in order to stabilizethe emulsion and facilitate its association with Apo E, and preferablyin the range of 0.4 to 0.5% (w/v) for hepatocyte-selective delivery ofiodinated triglycerides. In accordance with preferred embodiments of theinvention, the molar ratio of cholesterol to emulsifier, which may be anatural, synthetic, or semi-synthetic phospholipid, has been found todirectly affect the particle diameter and dimensional stability. Thepreferred molar ratio of cholesterol to phospholipid for achieving anemulsion which successfully mimics chylomicron remnants is in the rangeof 0.05 to 0.70, and more specifically at 0.40 for hepatocyte-selectivedelivery of iodinated triglycerides.

"Hepatocyte-selectivity" as the term is used herein can be demonstratedby a liver to spleen uptake ratio of >4:1 at 30 minutes after injectionbased on % dose/organ values (see, Example 2 below) and biliaryclearance and elimination profiles (see, FIGS. 11 and 13-16). Based onthe entire body of scientific literature describing the anatomicalfeatures of the hepatocytes and the surrounding sinusoidal spaces,particles with a diameter greater than approximately 300 nm are unableto access the surface of the hepatocytes. A hepatocyte-selective vehicleprimarily delivers compound intracellularly to the targeted tissue anddoes not significantly deliver compound nonspecifically to the surfaceof the cells (extracellular) or to the sinusoids between the cells (see,FIGS. 17 and 18).

In a composition aspect of the invention, the synthetic heat stablehepatocyte-selective oil-in-water emulsion of the present invention hasthe general formula:

1. up to 50% (w/v) lipophilic core components;

2. up to 10% emulsifier (w/v);

3. up to 5% cholesterol (w/v);

4. up to 5% osmolality adjusting agent (w/v);

5. optionally, up to 5% antioxidant (w/v); and

6. sterile, distilled water to final volume.

The types of agents which can be delivered to the hepatocytes byincorporation into the lipophilic core of the synthetic oil-in-wateremulsions of the present invention are lipophilic and/or amphipathiccompounds, which may be bioactive or bioinactive. The lipophilic corecomponents comprise up to 50% (w/v) of the emulsion, and preferablybetween about 10% and 30% (w/v). The lipophilic core may comprise anypharmaceutically acceptable fat or oil of natural, synthetic, orsemi-synthetic origin which is a pharmacologically inert nonpolar lipidthat will locate in the lipophilic core of the oil-in-water emulsion.Specific examples include, without limitation, triglycerides,illustratively, triolein, a naturally-occurring triglyceride of highpurity (available from Sigma Chemical Company, St. Louis, Mo.), or oilsof animal or vegetable origin, such as soybean oil, safflower oil,cottonseed oil, fish oils, and the like.

In other preferred embodiments, the lipophilic core includes lipophilicor amphipathic bioactive agents which may be used for diagnostic ortherapeutic purposes. For diagnostic purposes, exemplary agents include,but are not limited to, halogenated triglycerides, such as iodinated orfluorinated triglycerides, which may contain a stable or radioactiveisotope of the halogen.

In particularly preferred embodiments, the lipophilic core includes amixture of at least one pharmacologically inert oil and a bioactive orinactive agent in a molar ratio in the range of 0.3 to 2, and morepreferably 1:1. Preferably, the lipophilicity of each core component iscomparable to ensure suitable blending of the lipid components.

In iodinated embodiments, iodine-containing lipids, of the type known inthe art, can be used. Such lipids include iodinated fatty acids in theform of glycerol or alkyl esters. However, in particularly preferredembodiments, the iodine-containing lipids are synthetic aromaticcompounds of known purity which are stabilized against in vivodegradation of the iodine linkage. Illustrative examples of radioactiveor non-radioactive halogenated triglycerides useful in the practice ofthe invention include, without limitation, iodinated triglycerides ofthe type described in U.S. Pat. No. 4,873,075 issued on Oct. 10, 1989;U.S. Pat. No. 4,957,729 issued on Sep. 18, 1990; and U.S. Pat. No.5,093,043 issued on Mar. 3, 1992. Exemplary iodinated triglycerides are2-oleoylglycerol-1,3-bis[7-(3-amino-2,4,6-triiodophenyl)heptanoate](DHOG) and2-oleoylglycerol-1,3-bis[4-(3-amino-2,4,6-triiodophenyl)butanoate](DBOG).

Clinically, ¹²² I, ¹²³ I, ¹²⁵ I, and ¹³¹ I are the iodine isotopes mostoften used with currently available scanning instrumentation. Of course,¹³¹ I-radiolabeled triglycerides may be used for therapeutic purposes,as is known in the art. However, any radioactive isotope of iodine iswithin the contemplation of the invention. A listing of all iodineisotopes is available at pages B-303-B-305 of the Handbook of Chemistryand Physics, 38th edition, CRC Press, 1977-1978. It should be noted that¹²⁷ I is the naturally-occurring stable isotope and is not considered tobe "radioactive".

In fluorinated embodiments, specific examples include stable orradioactive (¹⁹ F) fluorinated triglycerides which are analogous to theiodinated triglycerides discussed above, illustrativelyglyceryl-2-oleoyl-1,3-bis(trifluoromethyl)phenyl acetate. In alternativeembodiments of the invention, fluorine-containing lipids may be estersor triglycerides of perfluoro-t-butyl-containing fatty acid compounds,such as described in U.S. Pat. No. 5,116,599 and 5,234,680,illustratively, 7,7,7-trifluoro-6,6-bis (trifluoromethyl)-heptanoic acidor 8,8,8-trifluoro-7,7-bis(trifluoromethyl)-octanoic acid. Of course,these examples are merely illustrative of the many specific examples oflipophilic or amphipathic fluorinated compounds suitable for use in thepractice of the invention, and are not in any way intended to beexclusive or limiting.

In still further embodiments of the invention, the bioactive agent maycomprise brominated compounds, such as brominated ethyl esters of fattyacids or monobrominated perfluorocarbons.

Potential therapeutic agents for inclusion in the lipophilic core of thesynthetic oil-in-water emulsion of the present invention includelipophilic derivatives of oligonucleotides or nucleic acids, orlipophilic and/or amphipathic derivatives of anti-cancer agents, such asesters of methotrexate, mitomycin C, fluorodeoxyuridine or doxorubicin.

The monolayer surrounding the nonpolar lipophilic core comprises up toabout 10% (w/v) of a polar lipid monolayer component, which may be anemulsifier. Phospholipids of natural, synthetic, or semi-syntheticorigin are suitable for use in the practice of the invention.Traditional lipid emulsions for delivery of bioactive agents use naturalphospholipids, such as soy lecithin and egg phosphatidylcholine (e.g.,Intralipid). In preferred embodiments of the present invention, theemulsion components are synthetic, semi-synthetic, and/or naturallyoccurring components of known origin, purity and relativeconcentrations. The improper use of egg lecithins (mixtures ofphospholipids) and/or crude oils (cottonseed, poppy seed, and the like)in prior art emulsions may result in variable and non-reproduciblecompositions.

In a specific advantageous embodiment, dioleoylphosphatidylcholine(DOPC) is used as an emulsifier, or monolayer surfactant. DOPC is asemi-synthetic, chemically defined phospholipid emulsifier of highpurity (available from Avanti Polar Lipids, Alabaster, Ala.). Of course,other surface active agents which are suitable for parenteral use can besubstituted for all or a portion of the polar lipid monolayer component.The naturally-occurring phospholipids are advantageous because thesephospholipids have a reasonable potential for interaction withapolipoproteins and an appropriate transition temperature, i.e., theyare in the liquid state at physiologic temperatures.

The osmolality of the emulsion is adjusted to 400-500 mOsm/kg with up to5% w/v of an osmolality adjusting agent, such as USP glycerol orglucose. The oil-in-water emulsion of the present invention produces adelivery vehicle which is nearly isotonic relative to blood. This is insharp contrast to most commonly used ionic contrast media which haveosmolalities 3 to 5 times higher than that of the blood which can resultin pain and tissue injury at the site of injection.

The remainder of the emulsion formulation comprises the bulk or aqueousphase. In the practice of a preferred embodiment of the presentinvention, the aqueous phase is sterile water of a grade suitable forparenteral administration. The inclusion of salt (NaCl) in the aqueousphase, such as by the use of 0.9% saline, results in emulsions whichhave a mean particle diameter as much as twice the size of salt-freeemulsions. Furthermore, the presence of salt in the formulation has anadverse effect on the ability of the emulsion to survive autoclavesterilization without a significant change in mean particle size as wellas on the temporal stability of an autoclaved emulsion.

In addition to NaCl, pH-adjusting agents frequently used in theformulation of parenteral emulsions, such as NaOH or the sodium salts ofmost aqueous buffers, introduce excessive sodium ions into the bulkphase. These agents have been found to have deleterious effects onparticle size, stability, and the ability to survive autoclaving.

Other conventional additives, such as antioxidants, buffers,preservatives, viscosity adjusting agents, and the like, may be includedin the composition. In particular, up to 5% w/v of an antioxidant, suchas α-tocopherol, flavinoids, BHT, or BHA, is recommended. However, theadditive should not adversely affect the physical characteristics of theemulsion, such as particle size or shelf and heat stability.

The techniques used to formulate the oil-in-water emulsions of thepresent invention are important in achieving small particle diameter,uniform size distribution, lack of liposome contamination, etc. all ofwhich contribute to hepatocyte-selectivity and heat stability.

In accordance with a method of making aspect of the invention, thelipophilic components of the oil-in-water emulsion including nonpolarcore lipids, polar lipid emulsifiers, and other lipophilic components,such as bioactive or bioinactive agents, are blended together to form apreblended lipid phase. The aqueous components are combined and added tothe preblended lipid phase. The preblended lipid phase and aqueouscomponents are homogenized to form a crude oil-in-water emulsion. Thecrude oil-in-water emulsion is subjected to ultra high energyemulsification to produce a fine oil-in-water emulsion having a meanparticle diameter of the oil phase between 50 to 200 nm with greaterthan 98% of the particles being less that 300 nm. In preferredembodiments of the invention, the fine oil-in-water emulsion issequentially filtered.

In a preferred method aspect of the invention, the lipid components areinitially blended or homogenized using a high speed mixer, such as aPolytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) or UltraTurrax (IKA-Works, Cincinnati, Ohio), operating at 12,500 rpm at 55° C.for at least 5 minutes. Then, the aqueous components are added to thepreblended lipid components and pre-emulsified by high speed mixing orhomogenization at 25,000 rpm under the same, or similar, conditions toform a crude emulsion. Final processing is accomplished with ultra highenergy mixing equipment, such as a MicroFluidizer high pressurehomogenizer (Model 110S, Microfluidics Corp., Newton, Mass.; see, USPN4,533,254), or equivalent equipment, such as the Emulsiflex (AvestinInc., Ottawa, Ontario, Canada) or the Manton-Gaulin (APV Gaulin Rannie,St. Paul, Minn.), operating in the recycling mode at 30-60° C. and10,000 to 30,000 psi, and preferably at about 12,500-18,200 psi, for upto about 20 minutes. After processing, the emulsion is passedsequentially through sterile 0.45 μm and 0.22 μm sterile filters. Thesequential filtration removes any large particles and partiallysterilizes the product.

The temperature for high energy mixing is illustrative, and should bechosen relative to the bioactive agent. In other words, the temperatureshould be greater than or equal to the transition temperature or meltingpoint of the bioactive agent. An upper bound, however, is determined bywhether the temperature would cause degradation or decomposition of anycomponents in the composition.

The use of an ultra high pressure homogenizer ensures small particlesize with a narrow size range distribution so that the resultingemulsion will simulate chylomicron remnants. Conventional systems forforming emulsions, such as homogenizers, sonicators, mills, and shakingsystems provide a shearing force on the liquid components whereas theultra high energy mixing equipment puts the emulsion components underpressure and forces them through small openings to reduce particle size.Size distribution may be measured by a Nicomp 370 Dynamic Laser LightScattering Autocorrelator (Nicomp Particle Sizing Systems, SantaBarbara, Calif.) or similar equipment. A lipid emulsion, which issuitable for the practice of the present invention, will have a meanparticle diameter less than about 300 nm, and preferably in the range of50 to 200 nm as measured by Nicomp number weighting analysis. Theparticles should have a narrow size distribution, with about 98% of theparticles being in the 50 to 300 nm. No particles should be detectedwith a diameter of greater than 1 μm.

The size distribution should be stable for a minimum shelf-life periodof ninety days. By "shelf-stable" is meant that the mean diameter of theemulsion particles should not change by greater than 15% at 90 dayspost-formulation, or more than 20% over the shelf-life. The emulsion canbe stored for up to 2 years at room temperature. Under ideal conditions,the product is stored under nitrogen at 4° to 8° C. and is, preferably,protected from light.

In preferred embodiments, the emulsion is sterilizable by heat or coldfiltration, for example. Standard or intermittent autoclavingtechniques, such as 20 minutes exposure to pressurized steam at 121° C.,should not adversely affect the size distribution of the emulsion. Asused herein the term "heat stable" means that the emulsion has theability to withstand standard heat sterilization (e.g., 20 minutesexposure to steam at 121° C.) without a significant change in meanparticle size distribution. A significant feature of thecomposition/formulation process of the present invention is the abilityof the emulsion to retain its ability to associate apo E followingautoclaving. Therefore, the term "heat stable" as used herein to definethe microemulsion of the present invention includes the ability toassociate with apo E after heat sterilization (see, Table 3).

In a method of use aspect of the invention, an oil-in-water emulsion ofthe present invention containing a contrast enhancing agent isadministered to a mammal and the mammal is subjected to x-ray computedtomographic imaging after the emulsion has reached the target site. Inalternative methods of use, appropriate oil-in-water emulsions,containing bioactive agents suitable for other diagnostic modalities,such as proton magnetic resonance imaging (MRI), ¹⁹ F-MRI, orscintigraphy may be administered for visualization and/or detection. Instill another method of use embodiment, a therapeutic agent, such as a¹³¹ I-containing triglyceride, is delivered to a target site in anoil-in-water emulsion of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Comprehension of the invention is facilitated by reading the followingdetailed description, in conjunction with the annexed drawing, in which:

FIG. 1 is a diagrammatic representation of an oil phase particle of theoil-in-water emulsion of present invention;

FIG. 2 is an illustrative preparatory scheme for a series of fluorinatedor iodinated triglycerides, specifically 1,3-disubstitutedtriacylglycerols, suitable for use in the practice of the presentinvention;

FIG. 3 is a graphic representation of a typical distribution of the oilphase particle size, in nm, in an emulsion of the present invention asmeasured by Nicomp Number Weighting analysis;

FIG. 4 is a graphic representation of a typical distribution of the oilphase particle size, in nm, in an emulsion of the present invention asmeasured by Nicomp Intensity Weighting analysis;

FIG. 5 is a graphic representation of the mean particle diameter (nm)for microemulsions containing 10% core lipids (w/v), including 5%triolein and 5% iodinated triglyceride (DHOG), 2.4% (w/v) phospholipid(DPOC) and varying amounts of cholesterol, so that the molar ratio ofcholesterol to phospholipid ranges from 0.3 to 0.69;

FIG. 6 is a graphic representation of the mean particle diameter (nm) asa function of time (days) post-formulation of microemulsions of the typeshown in FIG. 5 and containing no cholesterol or cholesterol in a molarratio of cholesterol to phospholipid of 0.1 or 0.4;

FIG. 7 is a graphic representation of the mean particle diameter (nm) ofthe microemulsion shown in FIG. 6 (CH:PC=0.4) as a function of timebefore and after steam sterilization in an autoclave;

FIG. 8 is an axial CT image (3 mm slice thickness) of a tumor-bearingrat (Morris Hepatoma 7777) taken at 60 minutes after administration of aCT contrast producing agent which is an oil-in-water emulsion inaccordance with the present invention;

FIG. 9 is an axial CT image (3 mm slice thickness) of tumor-bearing rat(Morris Hepatoma 7777) taken one hour after administration of a CTcontrast producing agent which is an oil-in-water emulsion in accordancewith the present invention;

FIG. 10 is a graphic representation of the tissue biodistributionprofile in liver (▪) and plasma (∘) expressed as tissue concentration (%administered dose per organ) as a function of time (hrs) followinginjection of a radioiodinated oil-in-water emulsion of the presentinvention;

FIG. 11 is a graphic representation of the cumulative fecal and urinaryelimination of radioactivity expressed as % administered dose as afunction of time (hrs) following injection of a radioiodinatedoil-in-water emulsion of the present invention;

FIG. 12 is a graphic representation of CT liver intensity followingintravenous bolus administration of a radioiodinated oil-in-wateremulsion of the present invention expressed as image intensity inHounsfield Units (HU) as a function of time (days) followingadministration;

FIGS. 13 to 16 are CT images of the hepatobiliary system of a dog taken24 hours after administration of a CT contrast agent which is anoil-in-water emulsion in accordance with the present invention;

FIGS. 17 and 18 are microscopic autoradiographs of liver tissue of acontrol animal and a test animal at 3 hours post-injection of a vehicle(control) or an oil-in-water emulsion containing a radioiodinatedtriglyceride in accordance with the present invention; and

FIG. 19 is a graphic representation of the elimination profile for aradioiodinated oil-in-water emulsion and/or its metabolites obtained bydirect cannulation of the bile duct and ureters of rats expressed ascumulative elimination (% dose) for biliary elimination (▪) and urinaryelimination (▾) and bile flow (; ml/min) as a function of time (mins)following injection the radioiodinated oil-in-water emulsion of thepresent invention.

DETAILED DESCRIPTION

FIG. 1 is a diagram of an oil phase particle 10 of the presentinvention. A lipophilic lipid core 12 is surrounded by a monolayer 11consisting of an emulsifier and cholesterol 13. The lipid core containsa pharmacologically inert fat or oil, such as a triglyceride (e.g.,triolein) and/or a lipophilic or amphipathic bioactive or bioinactiveagent, such as a radiologic agent. The polar moieties (spheres 14, e.g.,polar head portions of a phospholipid emulsifier) of the monolayer faceoutward into the bulk water phase (not specifically shown) whereas thenonpolar moieties (tails 15) of the monolayer are oriented toward thelipid core. A purely lipophilic compound to be delivered in accordancewith the principles of the invention would reside entirely in the coreof the lipid particle beneath the monolayer. On the other hand, anamphipathic compound, which has both lipophilic and hydrophiliccomponents, would reside in the particle by interaction with both thecore and the monolayer. For example, such an amphipathic agent mighthave a long lipophilic tail to tether a hydrophilic moiety to theparticle. The lipophilic tail could intermix with the lipophilic tailsof the phospholipid/cholesterol components of the monolayer or couldextend into the core. FIG. I also shows large amorphous structures 16which represent apolipoproteins.

The following specific examples illustrate some of the many possibleoil-in-water emulsions which can be made in accordance with theprinciples of the invention.

Iodine Embodiments:

Illustrative examples of radioactive or non-radioactive polyhalogenatedtriglycerides particularly suitable for use in the practice of theinvention are described in U.S. Pat. No. 4,873,075 issued on Oct. 10,1989; U.S. Pat. No. 4,957,729 issued on Sep. 18, 1990; and U.S. Pat. No.5,093,043 issued on Mar. 3, 1992, the disclosures of which areincorporated by reference herein in their entirety. The iodinatedarylaliphatic triglyceride analogs of the aforementioned patents have atriglyceride backbone structure which is 1,3-disubstitutecl or1,2,3-trisubstituted with a 3-substituted 2,4,6-triiodophenyl aliphaticchain or a monoiodophenyl aliphatic chain. In preferred embodiments, allof the aliphatic chains, whether on the iodinated moiety or an openposition on the triglyceride backbone structure, are saturated orunsaturated aliphatic hydrocarbon chains of the type found innaturally-occurring fatty acids. Naturally-occurring fatty acids mayinclude those containing about 4-20 carbons, illustratively palmiticacid (16), palmitoleic acid (16:1), oleic acid (18:1), linoleic acid(18:2), arachidonic acid (20:4), etc.

Specific examples include, but are not limited to:glyceryl-2-palmitoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)iopanoate;glyceryl-2-palmitoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)dodecanoate;glyceryl-2-palmitoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)acetate;glyceryl-2-palmitoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)propionoate;glyceryl-1,2,3-triiopanoateglyceryl-1,2,3-tri-12-(3-amino-2,4,6-triiodophenyl)dodecanoate;glyceryl-1,3-di-17-(3-amino-2,4,6-triiodophenyl)heptadecanoate;glyceryl-1,2,3-tri-3-(3-amino-2,4,6-triiodophenyl)propionate;glycerol-2-palmitoyl-1,3-di-15-(p-iodophenyl)pentadecanoate;glyceryl2-oleoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)-butyrate;glyceryl-2-oleoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)-pentanoate,glyceryl 2-oleoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)-hexanoate;glyceryl 2-oleoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)-octanoate;glyceryl 2-oleoyl-1,3-di-(3-amino-2,4,6-triiodophenyl)-heptwioate, etc.

For the studies reported herein, iodinated triglycerides weresynthesized and radioiodinated with ¹²⁵ I via isotope exchange in a meltof pivalic acid in accordance with a method known in the art. Of course,radioiodination of the iodinated triglycerides, or one of theintermediates in their synthesis pathway, can be accomplished by avariety of techniques, known to those of skill in the art.

EXAMPLE 1

A series of iodinated triglycerides of the following general formula,specifically 1,3-disubstituted triacylglycerols, which are identified asCompounds 1-7 in Table 1, were synthesized in accordance with theillustrative preparatory scheme shown on FIG. 2. ##STR1##

                  TABLE 1                                                         ______________________________________                                        Compound n      X       Chemical Name                                         ______________________________________                                        1        0      H       2-oleoylglycerol-1,3-[bis-(3-amino-                                           2,4,6-triiodophenyl)acetate]                          2        1      H       2-oleoylglycerol-1,3-[bis-(3-amino-                                           2,4,6-triiodophenyl-propionate]                       3        1      ethyl   2-oleoylglycerol-1,3-bis[iopanoate]                   4        2      H       2-oleoylglycerol-1,3-bis[4-(3-amino-                                          2,4,6-triiodophenyl)butyrate]                         5        3      H       2-oleoylglycerol-1,3-bis[5-(3-amino-                                          2,4,6-triiodophenyl)pentanoate]                       6        4      H       2-oleoylglycerol-1,3-bis[6-(3-amino-                                          2,4,6-triiodophenyl)hexanoate]                        7        5      H       2-oleoylglycerol-1,3-bis[7-(3-amino-                                          2,4,6-triiodophenyl)heptanoate]                       ______________________________________                                    

Refering to FIG. 2, a general reaction scheme is shown for a series ofjodinated or fliorinated trilycerides, specifically 1,3-disubstitutedtriacyiglycerols suitable for use in the practice of the presentinvention (Compounds 22). In the illustrative embodiments of Example 1,compounds 22 are2-oleoylglycerol-1,3-bis-[3-amino-2,4,6-triiodophenyl)alkanoates],designated as Compounds 1-7 on Table 1, which were synthesized viadicyclohexycarbodiimide/4-dimethylaminopyridine (DCC/DMAP) coupling of a2-monoolein (Compounds 21) with 2 equivalents of the correspondingω-(3-amino-2,4,6-triidophenyl)alkanoic acid (Compounds 20) as describedbelow.

Preparation of ω(3-amino-2,4,6-triiodolphenyl)alkanoic acids:

Synthesis of the ω-(3-amino-2,4,6-triiodophenyl)alkanoic acids(Compounds 20) was accomplished in a similar fashion to existingliterature procedures (see, for example, Weichert, et al., J. Med.Chem., Vol. 29, p. 1674 and 2457 (1986). lopanoic acid is commerciallyavaliable and was purchased from CTC Organics, Atlanta, Ga.

Preparation of2-oleoylglvcerol-1,3-bis-[3-amino-2,4,6-triiodophenvl)alkanoates]:

A rapidly stirred suspension of 2-monoolein (1,2,3-trihydroxypropane2-oleate; 1.0 equiv), the ω-(3-amino-2,4,6-triiodophenyl)alkanoic acids(2.05-2.1 equiv), and a catalytic amount of DMAP (0.1 equiv) inanhydrous CH₂ Cl₂ (5 ml/mmol of alcohol) was treated with DCC (1.1 equivto acid). The resulting mixture was stirred under N₂ overnight at roomtemperature, diluted with CH₂ Cl₂ and filtered to remove precipitateddicyclohexyl urea. The filtrate was washed with 0.5 N HCl, saturatedaqueous NaHCO₃, H₂ O, and brine, and then dried (MgSO₄). The solvent wasremoved in vacuo, and the remaining residue was purified by columnchromatography to afford the desired triacylglycerols.

Compound 1:2-oleoylglycerol-1,3-bisr[2-(3-amino-2,4,6-triiodophenyl)acetate]

Treatment of a mixture of 2-(3-amino-2,4,6-triiodophenyl)acetic acid(1.50 g, 2.8 mmol), 2-monoolein (481 mg, 1.35 mmol), and DMAP (37 mg) inanhydrous CH₂ Cl₂ (20 ml) with DCC (635 mg, 3.1 mmol) according to theprocedure described above gave a solid residue (1.56 g), which waspurified by column chromatography on silica gel (4.5×30 cm) eluted withhexanes/EtOAc/CHCl₃ (35:10:5) to give Compound 1 as a slightly yellowoil which resisted crystallization: yield 600 mg (32%); IR (CHCl₃) 3435,3325 (amine), 2900, 2830 (aliphatic CH), 1730 (ester C═O) cm⁻¹ ; ¹ H NMR(270 MHz, CDCl₃) 8.05 (s, 2H, aryl 5-H's), 5.4-5.2 (m, 3H, CH═CH,glycerol 2-H), 4.80 (s, 4H, NH₂), 4.32 (dd, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 4.24 (s, 4H, PhCH₂ 's), 4.15 (dd, 2H, glycerolOCH_(A) H_(B) CH(O)CH_(A) H_(B) O), 2.30 (m, 6H, CH₂ CO₂ and ═CHCH₂ 's),2.0-1.0 (m, 22H, CH₂ envelope), 0.88 (t, 3H, CH₃). Anal (C₃₇ H₄₈ O₆ N₂I₆) C, H.

Compound 2:2-oleovlglycerol-1,3-bis[3-(3-amino-2,4,6-triiodophenyl)propionate]

Treatment of a mixture of 3-(3-amino-2,4,6-triiodophenyl)propionicacid(1.16 g, 2.1 mmol), 2-monoolein (356 mg, 1.0 mmol), and DMAP (24 mg)in anhydrous CH₂ C₂ (15 ml) with DCC (444 mg, 2.15 mmol) according tothe procedure described above gave a residue (1.50 g), which waspurified by column chromatography on silica gel (4.5×30 cm) eluted withhexanes/EtOAc/CHCl₃ (16:2:1) to give Compound 2 as a slightly yellow oilwhich resisted crystallization: yield 1.30 g (92%); IR (CHCl₃) 3440,3330 (amine), 2905, 2830 (aliphatic CH), 1732 (ester C═O) cm⁻¹ ; ¹ H NMR(270 MHz, CDCl₃) 8.05 (s, 2H, aryl 5-H's), 5.4-5.2 (m, 3H, CH═CH,glycerol 2-H), 4.83 (s, 4H, NH₂ 's), 4.34 (dd, 2H, glycerol OCH_(A)H_(B) CH(O)CH_(A) H_(B) O), 4.15 (dd, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 3.38 (dt, 4H, PhCH₂ 's), 2.55 (dt, 4H, OC(O)CH₂'s), 2.35 (t, 6H, oleoyl CH₂ CO₂ and ═CHCH₂ 's), 1.99 (pst, 4H, CH₂ 's),1.29 (m, 22H, CH₂ envelope), 0.88 (t, 3H, CH₃). Anal (C₃₉ H₂₂ O₆ N₂ I₆)C, H.

Compound 3: 2-oleoylglycerol-1,3-bis[iopanoate]

Stirring a mixture of iopanoic acid (8.41 g, 14.7 mmol), 2-monoolein(2.50 g, 7.0 mmol), and DMAP (170 mg) in anhydrous CH₂ Cl₂ (70 ml) withDCC (3.33 g, 16.0 mmol) according to the procedure described above forthree days gave a residue (12.6 g), which was purified by columnchromatography on silica gel (10×23 cm) eluted with hexanes/EtOAc/CHCl₃(35:10:5) to give Compound 3 as a nearly colorless oil which resistedcrystallization: yield 5.37 g (53%); IR (CHCl₃) 3470, 3370 (amine),2930, 2860 (aliphatic CH), 1740 (ester C═O) cm⁻¹ ; ¹ H NMR (270 MHzCDCl₃) 8.07 (d, 2H, aryl 5-H's), 5.32 (m, 2H, CH═CH), 5.19 (m, 1H,glycerol CH), 4.85 (s, 4H, NH₂), 4.30 (m, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 4.10 (m, 2H, glycerol OCH_(A) H_(B) CH(O)CH_(A)H_(B) O), 3.32 (dq, 4H, PhCH₂ 's), 2.78 (m, 2H, iopanoyl CHCO₂), 2.28(t, 2H, oleoyl CH₂ CO₂), 2.05 (m, 4H, allylic CH₂ 's), 1.85 (m, 2H,iopanoyl CHCH_(A) H_(B) CH₃), 1.56 (m, 2H, iopanoyl CHCH_(A) H_(B) CH₃),1.4-1.2 (m, 20H, CH₂ envelope), 0.88 (t, 9H, CH₃). Anal (C₄₈ H₆₀ O₆ N₂I₆) C, H.

Compound 4:2-oleoylglycerol-1,3-bis[4-(3-amino-2,4,6-triiodophenyl)butanoate]

Stirring a mixture of 4-(3-amino-2,4,6-triiodophenyl)butanoic acid(7.00g, 12.6 mmol), 2-monoolein (2.19 g, 6.1 mmol), and DMAP (168 mg) inanhydrous CH₂ Cl₂ (80 ml) with DCC (2.85 g, 13.8 mmol) according to theprocedure described above for three days gave a residue (12.5 g), whichwas purified by column chromatography on silica gel (10×25 cm) elutedinitially with 1 liter of hexanes/EtOAc (5:1) and then withhexanes/EtOAc/CHCl₃ (75:15:10) to give Compound 4 as a slightly yellowoil which resisted crystallization: yield 5.08 g (58%); IR (CHCl₃) 3470,3370 (amine), 2930, 2860 (aliphatic CH), 1740 (ester C═O) cm⁻¹ ; ¹ H NMR(270 MHz CDCl₃) 8.04 (s, 2H, aryl 5-H's), 5.33 (m, 3H, CH═CH, andglycerol 2-H), 4.81 (s, 4H, NH2), 4.33 (m, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 4.20 (m, 2H, glycerol OCH_(A) H_(B) CH(O)CH_(A)H_(B) O), 3.06 (m, 4H, PhCH₂ 's), 2.48, (t, 4H, )O₂ CCH₂ 's), 2.33 (t,2H oleate O₂ CCH₂), 2.00 (m, 4H, allylic CH₂ 's), 1.84 (m, 4H, PhCH₂CH₂), 1.26 (d, CH₂ envelope), 0.89 (t, 3H, CH₃). Anal (C₄₆ H₅₆ O₆ N₂ I₆)C, H.

Compound 5:2-oleoylglycerol-1,3-bis[5-(3-amino-2,4,6-triiodophenyl)pentanoate]

DCC (635 mg, 3.1 mmol) was added to a stirred suspension of5-(3-amino-2,4,6-triiodophenyl)pentanoic acid(1.60 g, 2.8 mmol),2-monoolein (480 mg, 1.3 mmol), and DMAP (50 mg) in anhydrous CH₂ Cl₂(45 ml) according to the procedure described above for 36 hours.Following workup, a residue (12.5 g) was obtained, which was purified bycolumn chromatography on silica gel (4.6×42 cm) eluted withhexanes/EtOAc/CHCl₃ (80:15:5) to give Compound 5 as a slightly yellowoil which resisted crystallization: yield 1.35 g (71%); IR (CHCl₃) 3475,3375 (amine), 2940, 2850 (aliphatic CH), 1738 (ester C═O) cm⁻¹ ; ¹ H NMR(360 MHz, CDCl₃) 8.03 (s, 2H, aryl 5-H's), 5.26 (m, 3H, CH═CH, andglycerol 2-H), 4.79 (s, 4H, NH₂), 4.31 (m, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 4.16 (m, 2H, glycerol OCH_(A) H_(B) CH(O)CH_(A)H_(B) O), 3.02 (m, 4H, PhCHI₂ 's), 2.43 (t, 4H, O₂ CCH₂ 's), 2.30 (t,2H, oleate O₂ CCH₂), 2.01 (m, 4H, allylic CH₂ 's), 1.80 (m, 4H, PhCH₂CH₂ 's), 1.62-1.24 (m, CH₂ envelope), 0.86 (t, 3H, CH₃). Anal (C₄₃ H₆₀O₆ N₂ I₆) C, H.

Compound6:2-oleoylglycerol-1,3-bis[6-(3-amino-2,4,6-triiodophenyl)hexanoate]

DCC (801 mg, 3.9 mmol) was added to a stirred suspension of6-(3-amino-2,4,6-triiodophenyl)hexanoic acid(2.14 g, 3.7 mmol),2-monoolein (620 mg, 1.7 mmol), and DMAP (70 mg) in anhydrous CH₂ Cl₂(45 ml) according to the procedure described above for 48 hours.Following workup, a residue (3.26 g) was obtained, which was purified bycolumn chromatography on silica gel (3×25 cm) eluted withhexanes/EtOAc/CHCl₃ (80:15:5) to give Compound 6 as a slightly yellowoil which resisted crystallization: yield 2.04 g (81%); IR (CHCl₃) 3475,3375 (amine), 2940, 2850 (aliphatic CH), 1738 (ester C═O) cm⁻¹ ; ¹ H NMR(360 MHz, CDCl₃) 8.03 (s, 2H, aryl 5-H's), 5.30 (m, 3H, CH═CH, andglycerol 2-H), 4.79 (s, 4H, NH₂), 4.31 (m, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 4.16 (m, 2H, glycerol OCH_(A) H_(B) CH(O)CH_(A)H_(B) O), 3.01 (m, 4H, PhCH₂ 's), 2.34 (m, 6H, O₂ CCH₂ 's and oleate O₂CCH₂), 2.00 (m, 4H, allylic CH₂ 's), 1.72 (m, 4H, PhCH₂ CH₂ 's), 1.60,1.50, 1.27 (m, CH₂ envelope), 0.87 (t, 3H, CH₃). Anal (C₄₅ H₆₄ O₆ N₂ I₆)C, H.

Compound 7:2-oleoylglycerol-1,3-bis[7-(3-amino-2,4,6-triiodophenvl)heptanoate]

DCC (3.62 g, 17.5 mmol) was added to a stirred suspension of7-(3-amino-2,4,6-triiodophenyl)heptanoic acid(10.0 g, 16.7 mmol),2-monoolein (2.83 g, 7.9 mmol), and DMAP (180 mg) in anhydrous CH₂ Cl₂(120 ml) according to the procedure described above for 24 hours.Following workup, a residue (14.7 g) was obtained, which was purified bycolumn chromatography on silica gel (10×25 cm) eluted withhexanes/EtOAc/CHCl₃ (80:15:5) to give Compound 7 as a slightly yellowoil which resisted crystallization: yield 9.45 g (79%); IR (CHCl₃) 3450,3359 (amine), 2915, 2840 (aliphatic CH), 1740 (ester C═O) cm⁻¹ ; ¹ H NMR(360 MHz, CDCl₃) 8.03 (s, 2H, aryl 5-H's), 5.30 (m, 3H, CH═CH, andglycerol 2-H), 4.79 (s, 4H, NH₂), 4.31 (m, 2H, glycerol OCH_(A) H_(B)CH(O)CH_(A) H_(B) O), 4.16 (m, 2H, glycerol OCH_(A) H_(B) CH(O)CH_(A)H_(B) O), 3.00 (m, 4H, PhCH₂ 's), 2.32 (m, 6H, O₂ CCH₂ 's and oleate O₂CCH₂), 2.00 (m, 4H, allylic CH₂ 's), 1.72 (m, 4H, PhCH₂ CH₂ 's),1.61-1.26 (m, CH₂ envelope), 0.88 (t, 3H, CH₃). Anal (C₄₇ H₆₆ O₆ N₂ I₆)C, H.

The iodinated triglycerides of Example 1 were incorporated into thelipid core of an oil-in-water emulsion by formulation techniques inaccordance with the invention as set forth more completely in thefollowing examples.

EXAMPLE 2

In a specific illustrative embodiment, Compound 7 in Example 1, which isDHOG, was formulated into an oil-in-water emulsion for use as ahepatocyte-selective CT agent. The general formula is as follows:

    ______________________________________                                        10%        (w/v) Total Lipid                                                             triolein (TO) + DHOG                                                          TO:DHOG (w/w) = 1:1                                                2.4%       (w/v) phospholipid                                                            DOPC                                                               0.5%       (w/v) % cholesterol                                                           Cholesterol:DOPC (molar ratio) = 0.4                               5%         (w/v) USP glycerol                                                 0.6%       (w/v) α-tocopherol                                           sterile water as bulk aqueous phase                                           ______________________________________                                    

In terms of actual quantities, these concentrations and ratios translateto:

    ______________________________________                                        500.00 mg triolein                                                            500.00 mg DHOG                                                                 2.40 ml of 100 mg/ml DOPC in absolute ethanol                                 47.10 mg cholesterol                                                         500.00 mg USP glycerol                                                         61.90 mg α-tocopherol (added just after the cholesterol)               total to 10 ml sterile water                                                  ______________________________________                                    

The solid lipids and purified oils were weighed into a 50 ml glass tubefollowed by the addition of 2.40 ml of 100 mg/ml DOPC in ethanol and 5mls of ethyl acetate. The solvent was removed by evaporation undervacuum at 40° C. for about 10 minutes on a rotary evaporator. The tubewas removed from the rotary evaporator and 500.0 mg triolein were addedon top of the DOPC. 47.10 mg cholesterol were then added to the DOPC/TOmixture. Next, 500.0 mg DHOG were added to the lipid mixture. Finally,about 3 ml CHCl₃ were added to the tube to facilitate complete mixing ofthe lipids. Organic solvents, such as ethanol, ethyl acetate, orchloroform, are used only as necessary to introduce lipophilic,lipophobic, or amphipathic components into the emulsion.

The lipids were rinsed from the walls of the tube with 1 ml of ethylacetate:ethanol (2:1) and the tube was returned to the rotary evaporatorto remove the solvent under vacuum at 40° C. for about 30 minutes. Therotary evaporator was connected to a high vacuum line for another 40-45minutes.

A 500 mg aliquot of glycerol was added to the lipid mixture. Thecomponents were processed on the Polytron under a stream of nitrogen.Initial emulsification of this mixture was done at 12,500 rpm for 5minutes at less than 55° C. followed by the addition of the aqueousphase (about 6 ml sterile water). After the water was added, theemulsion was processed for 5 minutes at full speed (25,000 rpm) usingthe Polytron. The emulsion was rinsed from the generator with a smallvolume of sterile water and the contents were transferred to a gas-tightsyringe. A final volume of 10.0 ml emulsion was obtained by the additionof sterile water.

The emulsion was transferred to the MicroFluidizer 110S sample reservoirand the emulsion was processed at 34-36° C. for 10 minutes at 14,700 psiusing the continuous pass mode. Processed emulsion was collected fromthe unit, filtered through a 0.45 μm Acrodisc filter and then filteredthrough a 0.22 μm Acrodisc sterile filter directly into a sterilemultidose vial. The vial was stored overnight at room temperature priorto particle sizing.

In accordance with the present invention, the mean diameter of oil phaseparticles is between 50 and 200 nm (number weighted), with a narrowdistribution wherein no more than 2% of the particles have a diameterthat falls outside of the range (i.e., being greater than 300 nm).Typical distributions of the particle size in an emulsion of the presentinvention are shown graphically in FIG. 3 (Nicomp Number Weightinganalysis) and FIG. 4 (Nicomp Intensity Weighting analysis). The data arepresented in both Nicomp Intensity and Nicomp Number weighted formats toenable determination of not only the diameter at which most of theparticles exist (number weighting), but also the presence of small, butsignificant populations of large particles (intensity weighting). Thedistribution should not have any particles greater than 1 μm. Nor shouldthe emulsion contain more than a minimal amount of liposomes.

The molar ratio of cholesterol to emulsifier, which in this case isDOPC, directly affects the particle diameter. The preferred molar ratioof cholesterol to phospholipid for achieving an emulsion whichsuccessfully mimics chylomicron remnants is in the range of 0.05 to0.70, and more specifically at 0.40 for hepatocyte-selective delivery ofiodinated triglycerides as demonstrated in FIGS. 5 and 6.

FIG. 5 is a graphic representation of the mean particle diameter (nm) ofa 10% triglyceride emulsion containing 5% triolein (w/v) and 5% (wlv)iodinated triglyceride (DHOG), 2.4% (w/v) phospholipid (DPOC) andvarying amounts of cholesterol, so that the molar ratio of cholesterolto phospholipid ranges from 0.3 to 0.69. The microemulsions containing amolar ratio of 0.4 exhibited the smallest mean particle diameter (202.4nm), but the remaining microemulsions had particles sizes which wereacceptable.

FIG. 6 is a graphic representation of the mean particle diameter (nm) asa function of time (days) post-formulation for 10% emulsions containing5% triolein and 5% iodinated triglyceride (DHOG), 2.4% phospholipid(w/v) and varying amounts of cholesterol. Referring to FIG. 6, themicroemulsion containing a molar ratio of cholesterol to phospholipid of0.4 ("*") showed shelf stability over a 90 day test period whereas themicroemulsions containing no cholesterol ("▪") or a molar ratio ofcholesterol to phospholipid of 0.1 ("⋄") were not shelf stable. Thus,the appropriate amount of cholesterol contributes to shelf stability.

Cholesterol also stabilizes the monolayer to resist changes in diameterfollowing heat sterilization. FIG. 7 is a graphic representation of themean particle diameter (nm) of the emulsion of Example 2 as a functionof time before and after steam sterilization in an autoclave. The dataare expressed in both intensity and number weighted formats. Emulsionsprepared without cholesterol did not survive autoclave sterilization.

EXAMPLE 3

Biodistribution Studies

Radioactive emulsions of the iodinated triglyceride compounds of Example1 (see Table 1) were prepared by the technique set forth in Example 2.The microemulsions were administered intravenously (tail vein) to normalfemale Sprague-Dawley rats at a radiologic dose of 25-45 mg I/kg bodyweight for biodistribution studies. Following injection, groups ofanimals were sacrificed at various time points (30 minutes, 3 hours, and24 hours) and the appropriate tissues were removed, rinsed free ofblood, and analyzed for radioactivity. The liver, spleen, and bloodvalues are listed in Table 2 as the percent administered dose per organ±SEM (based on the actual organ weights for each animal, n=3, and theliterature values for the blood as a percent of total body weight).Other tissues, including kidney, lung, bone marrow, ovaries, adrenals,thyroid, heart, fat, and muscle generally contained low levels ofradioactivity.

                                      TABLE 2                                     __________________________________________________________________________    Cmpd.                                                                             1    2    3    4    5    6    7                                           __________________________________________________________________________    30 min                                                                        liver                                                                             69.8 ± 7.1                                                                      66.2 ± 4.9                                                                      78.0 ± 2.5                                                                      75.4 ± 3.6                                                                      65.1 ± 2.2                                                                      66.8 ± 2.4                                                                      67.7 ± 6                                 spleen                                                                            12.0 ± 1.1                                                                      13.0 ± 0.2                                                                      14.4 ± 0.8                                                                      13.7 ± 1.1                                                                       9.5 ± 0.8                                                                      13.9 ± 0.5                                                                      10.5 ± 2                                 blood                                                                              7.6 ± 2.3                                                                       5.1 ± 2.0                                                                       2.6 ± 0.8                                                                       5.8 ± 3.5                                                                       0.9 ± 0.3                                                                       4.8 ± 1.2                                                                       6.6 ± 3                                 3 hour                                                                        liver                                                                             79.0 ± 0.3                                                                      71.7 ± 0.6                                                                      80.0 ± 8.0                                                                      93.4 ± 4.3                                                                      60.7 ± 2.9                                                                      66.0 ± 1.9                                                                      45.8 ± 2                                 spleen                                                                            14.5 ± 1.2                                                                      12.9 ± 0.8                                                                      11.3 ± 1.4                                                                      11.7 ± 2.9                                                                       8.0 ± 0.4                                                                      11.3 ± 0.4                                                                       8.2 ± 1                                 blood                                                                              0.5 ± 0.1                                                                       0.7 ± 0.0                                                                       0.3 ± 0.0                                                                       0.9 ± 0.0                                                                       0.9 ± 0.7                                                                       2.2 ± 0.3                                                                       2.7 ± 0                                 24 hour                                                                       liver                                                                             72.5 ± 0.3                                                                      69.8 ± 1.7                                                                      77.0 ± 3.4                                                                      74.3 ± 1.6                                                                      36.1 ± 1.6                                                                      34.9 ± 3.4                                                                       9.6 ± 1                                 spleen                                                                            12.2 ± 1.2                                                                      11.2 ± 2.2                                                                      12.5 ± 1.0                                                                       8.8 ± 0.5                                                                       6.4 ± 0.3                                                                       5.8 ± 0.7                                                                       0.8 ± 0                                 blood                                                                              0.2 ± 0.0                                                                       0.5 ± 0.2                                                                       0.9 ± 0.1                                                                       1.5 ± 0.0                                                                       1.4 ± 0.1                                                                       1.1 ± 0.2                                                                       2.5 ± 0                                 __________________________________________________________________________

All seven analogs displayed a high degree of liver specificity asdemonstrated in the tissue distribution results reported in Table 2.From 65 to 78 percent of the radioactivity of the injected dose residedin the liver 30 minutes after administration. At 3 hours, from 46 to 93percent of the radioactivity remained in the liver. Liver to bloodratios at 3 hours ranged from 11 to 400 based on calculations ofinjected dose per gram of tissue. The longer chain analogs (Compounds5-7) appeared to have undergone some degree of metabolism and subsequentelimination from the liver by 3 hours. The shorter chain analogs(Compounds 1-4), on the other hand, appeared to be more resistant to invivo metabolism and elimination from the liver as indicated by arelatively small decrease in liver radioactivity at 3 hours as comparedto 24 hours. Thus, while liver uptake is primarily dependent on thecharacteristics of the emulsion vehicle, metabolism and subsequentclearance of the targeted triglyceride analog from the liver may beinfluenced, at least in part, by the chemical structure of thetriglyceride, including the alkyl chain length. Shorter chain analogsare more resistant to in vivo degradation in part because they arepoorer substrates for both hepatic lipase and lysomal acid hydrolase.

In vivo Imaging Studies

Oil-in-water emulsions containing Compounds 3 and 7 were administered atdose levels ranging from 20 to 70 mg I/kg body weight to normal rats andtumor-bearing rats (Morris Hepatoma 7777) for CT evaluation. Tumormorphology was verified by gross pathologic inspection.

In normal rats, both compounds displayed a rapid uptake into the liverso that liver densities increased from a baseline mean of 58 HU tonearly 100 HU by 60 minutes. By 3 hours, CT density was declining forCompound 7 (DHOG), but remained high for the sterically hinderedCompound 3. Liver density decreased slightly for Compound 3 by 24 hours,but had returned nearly to baseline for Compound 7.

The presence of hypodense liver tumors ranging in size from 1 to 15 mmin diameter were readily detected in CT studies of tumor-bearing rats(Morris Hepatoma 7777). Peak liver densities persisted for up to 2 hourswith Compound 7 and for over 24 hours with Compound 3. In both cases,the imaging characteristics were similar to or exceeded those observedfollowing administration of a urographic control (Omnipaque 300,Sanofi/Winthrop, New York, N.Y.; at a dose of 600 mgI/Kg body weight).However, the contrast agents of the present invention produced imageswith less than one-tenth the iodine dose.

FIG. 8 is an axial CT image (3 mm slice thickness) of one tumor-bearingrat 60 minutes after administration of the composition of Example 2.Since the tumor cells are physiologically distinct from the hepatocytes,the tumor cells do not enhance and appear hypointense relative to theenhanced hepatocytes. The contrast distinction makes identification andlocalization of small lesions considerably easier In this particularcase, the tumors are 4 mm in diameter. FIG. 9 is an axial CT scan ofanother tumor-bearing rat at one hour post-administration. The presenceof two tumors is clearly shown. The tumor on the left is 10 mm indiameter and the one on the right is 6 mm in diameter.

Metabolic/Elimination Studies

A lipid emulsion prepared in accordance with Example 2 (10% total corelipid content) was used in the tissue distribution and eliminationstudies reported hereinbelow. Fasted female Sprague-Dawley rats wereadministered radioactive (¹²⁵ I-ITG) emulsion at a dose of 25-35 mg I/kgby tail vein injection. The amount of radioactivity in 13 tissues wasdetermined as a function of time after injection. The results are shownin FIG. 10 which is a graphic representation of the tissuebiodistribution profile in liver and plasma. The tissue concentration as% administered dose per organ (mean ±SEM) in liver (▪) and plasma (∘)are plotted as a function of time (hrs) following injection.

As shown in FIG. 10, excellent liver-selective delivery of ITG wasobserved in tissue distribution studies using female rats. At 30 minutespost-injection, over 65% of the total dose was accumulated in the liver.Other tissues, typically contained less that 2% of the injected dose,although the spleen did accumulate nearly 12% of the total dose. By 24hours, less than 5% of the injected dose remained in the liver.

Radioactive (¹²⁵ I-ITG) emulsion was also administered to female ratsthat were subsequently placed in metabolic cages for 9 days. Feces andurine were collected daily and measured for radioactivity. Thecumulative fecal and urinary elimination of radioactivity, expressed as% administered dose, is plotted as a function of time (days) followinginjection of the radioactive emulsion in FIG. 11. Referring to FIG. 11,the lines are marked as follows: combined fecal and urinary elimination(▪), fecal elimination (), and urinary elimination (▾).

The results from the elimination studies show that the vast majority ofthe ITG was metabolized by the liver and eliminated through the biliarypathway. Fecal elimination comprised just over 80% of the totalelimination while urinary excretion contributed less than 20% of theelimination. In all, over 94% of the injected dose was recovered by theend of the experiment, with more than 80% being excreted by the secondday.

To assess the tissue-selective CT efficacy of the oil-in-water emulsionof the present invention, a fasted female dog was imaged at varioustimepoints up to 24 hours after injection of a bolus dose of theemulsion of Example 2. Enhancement of the liver paralleled the tissuedistribution profiles observed with rats. FIG. 12 is a graphicrepresentation of the CT image intensity of the liver (HU) plotted as afunction of time, in hours, following injection. As shown in FIG. 12,liver enhancement increased to a maximum by 60 minutes afteradministration and remained high for up to 2 hours. By 24 hourspost-injection, however, the liver intensity had returned topre-contrast values. The intensity of the gallbladder increased steadilywith time so that by 24 hours post-injection, the CT intensity hadincreased by 642% over pre-contrast values. The values plotted on FIG.12 represent the mean ±SEM of 10-15 ROI measurements from threesequential liver scans. Slice thickness for all scans was 5 mm.

FIGS. 13 to 16 are CT images at 24 hours post-administration. Referringto FIG. 13, the gall bladder, in the upper left corner, is greatlyenhanced. FIG. 14 shows an image at a lower anatomical level of the sameanimal at approximately the same time. The gall bladder is full andgreatly enhanced. Cholecystokinin was administered to the dog toinitiate emptying of the gall bladder. FIG. 15 is an image generated 5minutes post-administration of the cholecystokinin. The cystic andcommon bile duct are now opacified. FIG. 16 is a lower level slice ofthe same dog taken 20 minutes post-administration of thecholecystokinin. The contrast enhancement is now located in the bowel.Material that is eliminated from the hepatocytes into the bile iscollected and concentrated in the gall bladder. Therefore, DHOG and/orits metabolites that have been processed by the hepatocytes andeliminated in the bile will accumulate in the gallbladder. Both theliver tissue and the biliary system were enhanced on CT images followingadministration of DHOG in the oil-in-water lipid emulsion of the presentinvention.

The biodistribution studies demonstrated excellent target tissuespecificity, with the vast majority of the injected dose being localizedto the liver. Data from metabolic clearance studies suggest delivery ofthe ITG to the hepatocytes, as more than 80% of the injected dose israpidly eliminated in the feces through the bile. The hepatocytetargeting was also confirmed in CT imaging studies, where the contentsof the gallbladder showed tremendous increases in image intensity withtime as ITG accumulated in the gallbladder bile.

The hepatocyte-selective nature of the emulsions of the presentinvention permit imaging of the hepatobiliary system. Therefore, use ofthe emulsions would be particularly advantageous in the diagnosis and/ortreatment of any disease that alters the hepatic lipase and lysomal acidlipase activity, such as diabetes, cancer, cirrhosis, alcoholism,primary and metastatic liver tumors, hepatitis, cholecystitis,obstructive jaundice, liver transplant functional assessment, fibroticliver, fatty liver, and many others.

EXAMPLE 4

For comparative purposes, oil-in-water emulsions were formulated usingonly sonication (Formulation 4a) or high speed mixing on a Polytron plussonication (Formulation 4b). The components of Formulations 4a and 4bare as follows:

    ______________________________________                                               500.0 mg triolein                                                             506.6 mg DHOG                                                                  2.40 ml of 100 mg/ml DOPC in CHCl.sub.3                                       47.1 mg cholesterol                                                          500.0 mg USP glycerol                                                          11.9 mg α-tocopherol                                                   total to 10 ml sterile water                                           ______________________________________                                    

Formulation 4a:

2.40 ml of 100 mg/ml DOPC in a solvent, specifically CHCl₃, was added toa Corex tube. The solvent was evaporated under nitrogen until a residueformed in the bottom of the tube. 500 mg triolein were added on top ofthe DOPC and 47.1 mg cholesterol were added to the DOPC/TO mixture. Ananti-oxidant, 11.9 mg α-tocopherol, was added after the cholesterol.

In a separate small glass vial, 506.6 mg DHOG were dissolved inperoxide-free ether and transferred quantitatively to the lipid mixturein the Corex tube. Additional ether was added to the tube to facilitatecomplete mixing of the lipids. The lipids were heated gently under astream of nitrogen to drive off the ether. When most of the ether wasgone, the mixture was re-suspended in a small volume of additional etherand transferred to a gas-tight Hamilton syringe. The Corex tube wasrinsed with ether and the volume was added to the contents of thesyringe. About 5 ml total volume was obtained, 2 ml of which was theactual lipid component.

The syringe was arranged in a syringe pump set to deliver the contentsat a constant rate of 0.57 ml/min. Approximately 5.0 ml of anhydrousglycerol in sterile water were placed in a stoppered glass vial and amagnetic stir bar was used to mix the solution on a heating plate. Whenthe temperature of the aqueous glycerol solution was 37 to 40° C., thelipids were added to the vial through a polyethylene tube. The vial wasvented with a needle and exposed to a stream of nitrogen gas. At thespecified flow rate of 0.57 ml/min, addition of the lipids took about 10minutes. Constant stirring formed a crude emulsion. The tubing from thesyringe was removed after all traces of ether were gone and the vial wassealed under nitrogen. The sealed emulsion was allowed to mix underconstant, vigorous stirring for 1 hour at 37 to 40° C.

The crude emulsion was removed from the vial into a gas-tight syringeand the vial was rinsed twice with a small volume of anhydrous glycerol.The emulsion and the washes were combined in the syringe. Anhydrousglycerol was added to obtain a final volume of 10.0 ml and the emulsionwas mixed by inversion directly in the syringe.

The emulsion was separated into two separate and equal aliquots of 5 mlapiece. One of the 5 ml aliquots was subjected to emulsification bysonification. The crude emulsion was placed in a water bath at roomtemperature into which the horn of the sonicator was immersed. Thesonicator was set to run in continuous mode for 5 minutes at an outputsetting of 6. This setting resulted in a power output of 70-80% on theneedle dial of the sonicator. The emulsion was collected and stored in aglass-stoppered vial under nitrogen.

Formulation 4b:

The second 5 ml aliquot of crude emulsion was processed into a finalemulsion by homogenization with a Polytron homogenizer. The emulsion wasplaced in a glass Corex tube and homogenized, under nitrogen, for 5minutes at 25,000 rpm with a small volume Polytron generator.

EXAMPLE 5

An oil-in-water emulsion of the following composition was made using theformulation method of Example 2:

    ______________________________________                                               800 mg triolein                                                               200 mg DHOG                                                                   120 mg egg phosphatide                                                         10 mg cholesterol                                                            225 mg glycerol                                                               total volume to 10 ml with 0.9% saline                                 ______________________________________                                    

1.20 ml of 100 mg/ml egg phosphatide was added to a 50 ml glass tube.The solvent was evaporated under partial vacuum at 40° C. for about 45minutes on a rotary evaporator until the solvent was removed. The tubewas removed from the rotary evaporator and 800 mg triolein were added ontop of the egg phosphatide. 10 mg cholesterol were then added to theDOPC/TO mixture. Next, 200 mg DHOG were added to the lipid mixture.Finally, about 3 ml CHCl₃ were added to the tube to facilitate completemixing of the lipids.

The lipids were mixed by agitation and an additional 1 ml of CHCl₃ wasadded to rinse material from the walls of the tube. The lipid mixturewas returned to the rotary evaporator to remove the CHCl₃ under vacuumat 40° C. When the bulk of the solvent had been removed, the vacuumsource was switched to a direct drive vacuum pump for an additional 45minutes. The tube was then transferred from the rotary evaporator to ahigh vacuum line for another 1.5 hours. The tube was sealed, flushedwith nitrogen, and stored overnight at 8° C.

The tube and contents were warmed to 37° C. and connected to the highvacuum system to remove the final traces of solvent. A 225.6 mg aliquotof glycerol was then added to the lipid mixture and the components wereprocessed on the Polytron under a stream of nitrogen. Initialemulsification of this mixture was done at 12,500 rpm for 5 minutes atless than 55° C. followed by the slow addition of the aqueous phase.After the water was added, the emulsion was processed for 5 minutes at25,000 rpm using the standard Polytron generator. The emulsion wasrinsed from the generator with a small volume of sterile water and thecontents were transferred to a gas-tight syringe. A final volume of 10.0ml emulsion was obtained by the addition of water.

The emulsion was transferred to the MicroFluidizer 110S sample reservoirand the emulsion was processed for 10 minutes at 14,700 psi using thecontinuous pass mode. Processed emulsion was collected from the unit,filtered through a sterile 0.45 μm Acrodisc filter and then filteredthrough a 0.22 μm Acrodisc sterile filter directly into a sterile glassmultidose vial. The vial was stored under nitrogen.

EXAMPLE 6

Cholesterol not only helps to stabilize the monolayer to resist changesin diameter over time following heat sterilization, but facilitates theassociation of the emulsion with the apoproteins required for targetingthe emulsion to hepatocytes, particularly Apo E. Most importantly,however, emulsions made in accordance with the present invention willretain the ability to associate with apo E after heat sterilization asshown in Table 3 below. Emulsions formulated in Examples 2 to 5 wereincubated in lipoprotein-deficient plasma and re-isolated. Lowry assaywas used to determine the μg protein per ml emulsion since the amount ofprotein includes association with Apo AI and Apo AIV. Samples of theemulsions of Example 2 and to Example 5 were subjected to standardautoclaving and assayed for mean particle diameter.

                                      TABLE 3                                     __________________________________________________________________________                            μg protein                                         Emulsion                                                                           Initial Size                                                                            Size                                                                              Stable to                                                                          per ml                                                                             Apolipoprotein                                   Formula                                                                            NI/NN     Stability                                                                         Autoclave                                                                          emulsion                                                                           AI AIV                                                                              E C                                        __________________________________________________________________________    Ex. 4a                                                                             4.0-7.8 μm/182 nm                                                                    YES NO   3    -  ++ - -                                        Ex. 4b                                                                             350-764 nm/40-42 nm                                                                     YES YES  28   +  ++ - -                                        Ex. 5                                                                              213 nm/77 nm                                                                            YES NO   65   ++ +++                                                                              + -                                        Ex. 2                                                                              245 nm/80 nm                                                                            YES YES  78   ++ +++                                                                              + -                                        Ex. 5                                                                              6 μm/2.4 μm                                                                       YES      52   -  -  - -                                        autoclave                                                                     Ex. 2                                                                              272 nm/114 nm                                                                           YES      70   +++                                                                              +++                                                                              + -                                        Autoclave                                                                     __________________________________________________________________________     * C is Apo C II and C III                                                     Key:                                                                          - = not present                                                               + = present, low level                                                        ++ = present, medium level                                                    +++ = present, high level                                                

EXAMPLE 7

In another specific illustrative embodiment, DHOG (Compound 7 on Table 1in Example 1) was formulated into an oil-in-water emulsion having 20%total core lipid content for use as a hepatocyte-selective CT agent. Thegeneral formula is was follows:

    ______________________________________                                        20%        (w/v) Total Lipid                                                             Triolein (TO) + DHOG                                                          TO:DHOG (w/w) = 1:1                                                3.6%       (w/v) phospholipid                                                            DOPC                                                               0.5%       (w/v) % cholesterol                                                           Cholesterol:DOPC (molar ratio) = 0.4                               5%         (w/v) USP glycerol                                                 0.6%       (w/v) α-tocopherol                                           sterile water as bulk aqueous phase                                           ______________________________________                                    

In terms of actual quantities:

    ______________________________________                                        DHOG                 1000.3  mg                                               triolein             1002.7  mg                                               cholesterol          71.1    mg                                               α-tocopherol   61.2    mg                                               DOPC                 360     mg                                               glycerol             500.7   mg                                               sterile water        7.40    ml                                               ______________________________________                                    

The lipid core components were dissolved in a solvent which wassubsequently removed as set forth above in Example 2. USP glycerol wascombined with the lipid mixture and emulsified under nitrogen for 5minutes on the Polytron at 12,500 rpm. A 5.5 ml aliquot of sterile waterwas added and emulsification was continued at 25,000 rpm for 5 minutesat <55° C. The rough emulsion and generator rinse were combined andadjusted to 10 ml with sterile water prior to final emulsification inthe MicroFluidizer, Model 110-S, for 10 minutes at 14,700 psi between34.4-35.4° C. The emulsion was then passed through sterile filters of0.45 pm and 0.2 pm pore size into a sterile multidose vial.

EXAMPLE 8

In yet another specific embodiment, DBOG (Compound 4 on Table 1 inExample 1) was formulated into an oil-in-water emulsion having 20% totalcore lipid content, 3.6% DOPC, and a molar ratio of cholesterol to DOPCof 0.27. The general formula is as follows:

    ______________________________________                                        DBOG                 1000.5  mg                                               triolein             1004.3  mg                                               cholesterol          47.5    mg                                               α-tocopherol   66.2    mg                                               DOPC                 360     mg                                               glycerol             502.6   mg                                               sterile water        7.4     ml                                               ______________________________________                                    

The oil-in-water emulsion was processed was under the same conditionsdescribed in Example 7.

EXAMPLE 9

FIGS. 17 and 18 are photomicroscopic autoradiographs of liver tissue ofa control animal and a test animal, respectively. The autoradiographswere taken following injection of a TO emulsion (control) or anoil-in-water emulsion of the present invention (test) into a liveanimal, a three hour biodistribution period, and a subsequent liverperfusion and fixation process. The test oil-in-water emulsion used toobtain the autoradiographic image of FIG. 18 contained 10% w/v totallipid core content which was 5% radioiodinated2-oleoylglycerol-1,3-bis[iopanoate] (DIOG or Compound 3 from Table 1)and 5% TO in a formulation similar to Example 2. The control emulsioncontained 10% w/v TO in the lipid core and was used to assess whetherthe emulsion per se would cause morphologic damage to liver tissue.

FIG. 17 shows only the normal background radioactivity found in livingtissue and exhibits no morphologic damage. FIG. 18 showshepatocyte-selective delivery of the oil-in-water emulsion of thepresent invention, i.e., intracellular localization of the iodinatedtriglyceride. Referring to FIG. 18, grains of radioactivity associatedwith the iodinated triglyceride were mainly present within themorphological boundaries of hepatocytes as opposed to being localized atthe surface of the cell, located in liver sinusoidal spaces, or locatedin Kupffer cells. As such, the results of this autoradiographic studysupport the conclusion that the oil-in-water emulsion of the presentinvention delivers its contents primarily to the hepatocytes.Furthermore, the liver tissue of FIG. 18 exhibits no morphologic damagefrom the administration of the oil-in-water emulsion of the presentinvention.

EXAMPLE 10 Comparative Studies of Emulsions Comprising Pure ComponentsVersus Natural

In vivo CT imaging studies were conducted using soybean oil/eggphosphatide-based formulations and triolein/DOPC-based formulations inorder to assess the relative advantages of the use of pure, syntheticcompounds in the oil-in-water emulsions of the present invention.reliminary CT studies showed that soybean oil/egg phosphatideformulations performed essentially equally. However, CT imaging cannotdirectly distinguish between material delivered to the hepatocytes andmaterial delivered to the Kuppfer cells.

If evaluation of liver function is desired, however, a contrast agentmust be delivered to the hepatocytes, and not to the Kuppfer cells orother cells of the liver. With targeted delivery to the hepatocytes, therelative differences in initial enhancement of the liver, and subsequentclearance of the enhancement agent from the liver, can be observed by CTimaging and correlated to metabolic function. The biodistribution andelimination studies presented hereinbelow demonstrate that the soybeanoil/egg phosphatide-based formulations are not directed to thehepatocytes in the same manner as are the triolein/DOPC-basedformulations of the present invention.

Tissue biodistribution studies were conducted to evaluate iodinatedtriglyceride localization profiles with soybean oil/egg phosphatideformulations as compared to triolein/DOPC formulations. The soybeanoil/egg phosphatide formulation was chosen to simulate a typical priorart parenteral nutrition supplement such as Liposyn (AbbottLaboratories, North Chicago, Ill.) or Intralipid (Kabivitrum AB,Stockholm, Sweden). The composition of the soybean oil/egg phosphatideformulation used in the studies of Example 10 was as follows:

    ______________________________________                                        DHOG               500 mg                                                     soybean oil        500 mg                                                     egg phosphatide    120 mg                                                     glycerol           250 mg                                                     sterile water      to 10 ml final volume                                      ______________________________________                                    

An oil-in-water emulsion of the soybean oil/egg phosphatide wasprocessed in a manner analogous to Example 2. The triolein/DOPCformulation used in this study was identical to Example 2. Bothemulsions were formulated with a tracer amount of radiolabeled ITG andsubsequently administered to fasted female rats at a dose of 24-26 mgI/kg. The animals were sacrificed at various time points (30 minutes, 3hours, 24 hours) and 13 tissues were assayed for radioactivity. Theresults for the soybean oil/egg phosphatide formulation are shown belowin Tables 4A to 4C. The results for the triolein/DOPC formulation(Example 2) are shown below in Tables 4D to 4F.

                                      TABLE 4A                                    __________________________________________________________________________    30 Minutes:                                                                   10% DHOG Soybean Oil/Egg Phosphatide-LE                                                                      Mean                                                Mean     Mean     Mean    % dose/                                        Tissue                                                                             dpm/mg                                                                            SEM  % dose/g                                                                           SEM kg dose/g                                                                          SEM                                                                              organ                                                                              SEM                                       __________________________________________________________________________    Adrenal                                                                            16.250                                                                            1.898                                                                              0.283                                                                              0.040                                                                             0.057                                                                              0.007                                                                            0.015                                                                              0.002                                     Blood                                                                              6.919                                                                             0.656                                                                              0.119                                                                              0.008                                                                             0.024                                                                              0.002                                                                            1.208                                                                              0.114                                     Bone 23.141                                                                            2.188                                                                              0.400                                                                              0.038                                                                             0.082                                                                              0.008                                                                            0.283                                                                              0.027                                     Marrow                                                                        Fat  9.209                                                                             3.070                                                                              0.157                                                                              0.049                                                                             0.032                                                                              0.011                                                                            2.299                                                                              0.766                                     Heart                                                                              136.451                                                                           10.779                                                                             2.374                                                                              0.252                                                                             0.481                                                                              0.038                                                                            1.391                                                                              0.110                                     Kidney                                                                             5.122                                                                             0.120                                                                              0.089                                                                              0.002                                                                             0.018                                                                              0.000                                                                            0.137                                                                              0.003                                     Liver                                                                              653.371                                                                           23.129                                                                             11.296                                                                             0.275                                                                             2.304                                                                              0.082                                                                            75.842                                                                             0.667                                     Lung 78.456                                                                            13.129                                                                             1.357                                                                              0.232                                                                             0.277                                                                              0.046                                                                            1.549                                                                              0.260                                     Muscle                                                                             1.735                                                                             0.217                                                                              0.030                                                                              0.003                                                                             0.006                                                                              0.001                                                                            2.784                                                                              0.348                                     Ovary                                                                              7.939                                                                             2.651                                                                              0.138                                                                              0.047                                                                             0.028                                                                              0.009                                                                            0.010                                                                              0.003                                     Plasma                                                                             8.211                                                                             0.536                                                                              0.142                                                                              0.005                                                                             0.029                                                                              0.002                                                                            0.788                                                                              0.051                                     Spleen                                                                             440.404                                                                           111.704                                                                            7.515                                                                              1.687                                                                             1.553                                                                              0.394                                                                            3.715                                                                              0.872                                     Thyroid                                                                            29.459                                                                            7.809                                                                              0.507                                                                              0.134                                                                             0.104                                                                              0.028                                                                            0.008                                                                              0.002                                     __________________________________________________________________________

                                      TABLE 4B                                    __________________________________________________________________________    3 hours:                                                                      10% DHOG Soybean Oil/Egg Phosphatide-LE                                                                      Mean                                                Mean     Mean     Mean    % dose/                                        Tissue                                                                             dpm/mg                                                                            SEM  % dose/g                                                                           SEM kg dose/g                                                                          SEM                                                                              organ                                                                              SEM                                       __________________________________________________________________________    Adrenal                                                                            7.705                                                                             1.650                                                                              0.139                                                                              0.031                                                                             0.027                                                                              0.006                                                                            0.007                                                                              0.001                                     Blood                                                                              10.234                                                                            0.519                                                                              0.184                                                                              0.009                                                                             0.036                                                                              0.002                                                                            1.786                                                                              0.091                                     Bone 12.887                                                                            1.699                                                                              0.232                                                                              0.032                                                                             0.045                                                                              0.006                                                                            0.157                                                                              0.021                                     Marrow                                                                        Fat  5.100                                                                             0.801                                                                              0.091                                                                              0.013                                                                             0.018                                                                              0.003                                                                            1.273                                                                              0.201                                     Heart                                                                              101.935                                                                           13.810                                                                             1.831                                                                              0.249                                                                             0.359                                                                              0.049                                                                            1.038                                                                              0.141                                     Kidney                                                                             11.143                                                                            1.017                                                                              0.200                                                                              0.016                                                                             0.039                                                                              0.004                                                                            0.299                                                                              0.027                                     Liver                                                                              545.082                                                                           23.030                                                                             9.781                                                                              0.316                                                                             1.922                                                                              0.082                                                                            65.162                                                                             2.560                                     Lung 49.854                                                                            6.725                                                                              0.893                                                                              0.110                                                                             0.176                                                                              0.024                                                                            0.984                                                                              0.133                                     Muscle                                                                             1.866                                                                             0.336                                                                              0.033                                                                              0.006                                                                             0.007                                                                              0.001                                                                            2.994                                                                              0.539                                     Ovary                                                                              4.617                                                                             0.252                                                                              0.083                                                                              0.005                                                                             0.016                                                                              0.001                                                                            0.006                                                                              0.000                                     Plasma                                                                             10.039                                                                            0.752                                                                              0.180                                                                              0.014                                                                             0.035                                                                              0.003                                                                            0.963                                                                              0.072                                     Spleen                                                                             290.358                                                                           49.155                                                                             5.233                                                                              0.926                                                                             1.023                                                                              0.173                                                                            2.768                                                                              0.322                                     Thyroid                                                                            163.598                                                                           45.812                                                                             2.952                                                                              0.858                                                                             0.577                                                                              0.161                                                                            0.043                                                                              0.012                                     __________________________________________________________________________

                                      TABLE 4C                                    __________________________________________________________________________    24 hours:                                                                     10% DHOG Soybean Oil/Egg Phosphatide-LE                                                                      Mean                                                Mean     Mean     Mean    % dose/                                        Tissue                                                                             dpm/mg                                                                            SEM  % dose/g                                                                           SEM kg dose/g                                                                          SEM                                                                              organ                                                                              SEM                                       __________________________________________________________________________    Adrenal                                                                            4.844                                                                             0.506                                                                              0.103                                                                              0.011                                                                             0.021                                                                              0.002                                                                            0.005                                                                              0.001                                     Blood                                                                              6.210                                                                             1.018                                                                              0.131                                                                              0.021                                                                             0.027                                                                              0.004                                                                            1.346                                                                              0.220                                     Bone 9.066                                                                             3.160                                                                              0.193                                                                              0.069                                                                             0.040                                                                              0.014                                                                            0.138                                                                              0.048                                     Marrow                                                                        Fat  4.367                                                                             0.525                                                                              0.093                                                                              0.012                                                                             0.019                                                                              0.002                                                                            1.354                                                                              0.163                                     Heart                                                                              10.300                                                                            2.620                                                                              0.219                                                                              0.058                                                                             0.045                                                                              0.011                                                                            0.130                                                                              0.033                                     Kidney                                                                             10.659                                                                            0.677                                                                              0.226                                                                              0.016                                                                             0.047                                                                              0.003                                                                            0.355                                                                              0.023                                     Liver                                                                              195.966                                                                           33.186                                                                             4.142                                                                              0.684                                                                             0.858                                                                              0.145                                                                            33.451                                                                             4.592                                     Lung 19.644                                                                            1.547                                                                              0.415                                                                              0.027                                                                             0.086                                                                              0.007                                                                            0.482                                                                              0.037                                     Muscle                                                                             0.992                                                                             0.090                                                                              0.021                                                                              0.002                                                                             0.004                                                                              0.000                                                                            1.976                                                                              0.178                                     Ovary                                                                              3.151                                                                             0.076                                                                              0.067                                                                              0.002                                                                             0.014                                                                              0.000                                                                            0.005                                                                              0.000                                     Plasma                                                                             5.862                                                                             0.937                                                                              0.124                                                                              0.019                                                                             0.026                                                                              0.004                                                                            0.698                                                                              0.11l                                     Spleen                                                                             93.235                                                                            25.380                                                                             1.962                                                                              0.509                                                                             0.408                                                                              0.111                                                                            0.870                                                                              0.198                                     Thyroid                                                                            466.549                                                                           38.884                                                                             9.860                                                                              0.733                                                                             2.043                                                                              0.168                                                                            0.153                                                                              0.013                                     __________________________________________________________________________

                                      TABLE 4D                                    __________________________________________________________________________    30 Minutes:                                                                   10% DHOG-TO/DOPC LE                                                                                           Mean                                               Mean      Mean     Mean    % dose/                                       Tissue                                                                             dpm/mg                                                                             SEM  % dose/g                                                                           SEM kg dose/g                                                                          SEM                                                                              organ                                                                              SEM                                      __________________________________________________________________________    Adrenal                                                                            278.662                                                                            77.937                                                                             3.228                                                                              0.903                                                                             0.709                                                                              0.202                                                                            0.181                                                                              0.051                                    Blood                                                                              31.525                                                                             5.414                                                                              0.365                                                                              0.063                                                                             0.080                                                                              0.014                                                                            3.962                                                                              0.708                                    Bone 29.753                                                                             2.206                                                                              0.345                                                                              0.026                                                                             0.075                                                                              0.006                                                                            0.261                                                                              0.019                                    Marrow                                                                        Fat  9.657                                                                              0.691                                                                              0.112                                                                              0.008                                                                             0.024                                                                              0.002                                                                            1.731                                                                              0.118                                    Hear 89.151                                                                             8.516                                                                              1.033                                                                              0.099                                                                             0.226                                                                              0.022                                                                            0.653                                                                              0.065                                    Kidney                                                                             11.440                                                                             0.590                                                                              0.132                                                                              0.007                                                                             0.029                                                                              0.001                                                                            0.220                                                                              0.011                                    Liver                                                                              776.875                                                                            44.460                                                                             8.998                                                                              0.515                                                                             1.966                                                                              0.095                                                                            62.083                                                                             2.950                                    Lung 70.810                                                                             4.768                                                                              0.820                                                                              0.055                                                                             0.179                                                                              0.012                                                                            1.004                                                                              0.066                                    Muscle                                                                             3.003                                                                              0.195                                                                              0.035                                                                              0.002                                                                             0.008                                                                              0.000                                                                            3.461                                                                              0.225                                    Ovary                                                                              20.441                                                                             3.032                                                                              0.237                                                                              0.035                                                                             0.052                                                                              0.008                                                                            0.018                                                                              0.003                                    Plasma                                                                             43.216                                                                             8.366                                                                              0.501                                                                              0.097                                                                             0.110                                                                              0.022                                                                            2.985                                                                              0.598                                    Spleen                                                                             2446.667                                                                           265.950                                                                            28.338                                                                             3.080                                                                             6.206                                                                              0.714                                                                            15.876                                                                             1.622                                    Thyroid                                                                            95.207                                                                             22.890                                                                             1.103                                                                              0.265                                                                             0.240                                                                              0.056                                                                            0.018                                                                              0.004                                    __________________________________________________________________________

                                      TABLE 4E                                    __________________________________________________________________________    3 hours:                                                                      10% DHOG-TO/DOPC LE                                                                                           Mean                                               Mean      Mean     Mean    % dose/                                       Tissue                                                                             dpm/mg                                                                             SEM  % dose/g                                                                           SEM kg dose/g                                                                          SEM                                                                              organ                                                                              SEM                                      __________________________________________________________________________    Adrenal                                                                            60.237                                                                             9.900                                                                              0.704                                                                              0.134                                                                             0.152                                                                              0.025                                                                            0.039                                                                              0.006                                    Blood                                                                              29.681                                                                             2.452                                                                              0.344                                                                              0.025                                                                             0.075                                                                              0.007                                                                            3.717                                                                              0.322                                    Bone 24.467                                                                             6.187                                                                              0.280                                                                              0.064                                                                             0.062                                                                              0.016                                                                            0.215                                                                              0.055                                    Marrow                                                                        Fat  8.610                                                                              1.091                                                                              0.099                                                                              0.010                                                                             0.022                                                                              0.003                                                                            1.543                                                                              0.202                                    Heart                                                                              44.156                                                                             12.434                                                                             0.509                                                                              0.135                                                                             0.112                                                                              0.032                                                                            0.323                                                                              0.092                                    Kidney                                                                             21.658                                                                             0.540                                                                              0.251                                                                              0.001                                                                             0.055                                                                              0.002                                                                            0.416                                                                              0.012                                    Liver                                                                              485.278                                                                            54.873                                                                             5.623                                                                              0.629                                                                             1.226                                                                              0.135                                                                            37.574                                                                             3.887                                    Lung 44.880                                                                             0.558                                                                              0.521                                                                              0.017                                                                             0.113                                                                              0.002                                                                            0.636                                                                              0.009                                    Muscle                                                                             2.945                                                                              0.348                                                                              0.034                                                                              0.003                                                                             0.007                                                                              0.001                                                                            3.391                                                                              0.415                                    Ovary                                                                              20.164                                                                             5.997                                                                              0.231                                                                              0.064                                                                             0.051                                                                              0.015                                                                            0.018                                                                              0.005                                    Plasma                                                                             24.940                                                                             3.993                                                                              0.287                                                                              0.039                                                                             0.063                                                                              0.010                                                                            1.717                                                                              0.282                                    Spleen                                                                             1437.084                                                                           76.326                                                                             16.646                                                                             0.775                                                                             3.635                                                                              0.208                                                                            8.724                                                                              0.613                                    Thyroid                                                                            266.971                                                                            66.535                                                                             3.116                                                                              0.831                                                                             0.675                                                                              0.169                                                                            0.051                                                                              0.013                                    __________________________________________________________________________

                                      TABLE 4F                                    __________________________________________________________________________    24 hours:                                                                     10% DHOG-TO/DOPC LE                                                                                           Mean                                               Mean      Mean     Mean    % dose/                                       Tissue                                                                             dpm/mg                                                                             SEM  % dose/g                                                                           SEM kg dose/g                                                                          SEM                                                                              organ                                                                              SEM                                      __________________________________________________________________________    Adrenal                                                                            6.893                                                                              1.857                                                                              0.162                                                                              0.043                                                                             0.035                                                                              0.009                                                                            0.009                                                                              0.002                                    Blood                                                                              6.207                                                                              3.675                                                                              0.148                                                                              0.089                                                                             0.031                                                                              0.018                                                                            1.544                                                                              0.913                                    Bone 3.611                                                                              0.354                                                                              0.085                                                                              0.009                                                                             0.018                                                                              0.002                                                                            0.063                                                                              0.006                                    Marrow                                                                        FaI  2.489                                                                              0.474                                                                              0.059                                                                              0.011                                                                             0.013                                                                              0.002                                                                            0.887                                                                              0.172                                    Heart                                                                              4.226                                                                              0.891                                                                              0.100                                                                              0.022                                                                             0.021                                                                              0.005                                                                            0.061                                                                              0.013                                    Kidney                                                                             5.065                                                                              0.277                                                                              0.120                                                                              0.008                                                                             0.025                                                                              0.001                                                                            0.194                                                                              0.011                                    Liver                                                                              54.295                                                                             6.663                                                                              1.282                                                                              0.165                                                                             0.273                                                                              0.033                                                                            14.077                                                                             0.739                                    Lung 7.053                                                                              1.588                                                                              0.167                                                                              0.038                                                                             0.035                                                                              0.008                                                                            0.198                                                                              0.044                                    Muscle                                                                             0.635                                                                              0.059                                                                              0.015                                                                              0.001                                                                             0.003                                                                              0.000                                                                            1.453                                                                              0.140                                    Ovary                                                                              2.592                                                                              0.200                                                                              0.061                                                                              0.006                                                                             0.013                                                                              0.001                                                                            0.005                                                                              0.000                                    Plasma                                                                             2.396                                                                              0.108                                                                              0.056                                                                              0.002                                                                             0.012                                                                              0.001                                                                            0.328                                                                              0.016                                    Spleen                                                                             111.586                                                                            15.137                                                                             2.628                                                                              0.345                                                                             0.561                                                                              0.074                                                                            1.510                                                                              0.136                                    Thyroid                                                                            579.753                                                                            109.374                                                                            13.738                                                                             2.770                                                                             2.916                                                                              0.550                                                                            0.219                                                                              0.041                                    __________________________________________________________________________

Referring to Table 4A, the average accumulation of iodinatedtriglyceride in the liver, at 30 minutes post-injection, was 76% of theinjected dose per organ. Less than 4% of the total dose was present inthe spleen, and the blood contained less than 2% of the injected dose.All other tissues accumulated less that 2.5% of the administered dose.However, at 3 hours post-injection (Table 4B), the iodinatedtriglyceride delivered in the soybean oil/egg phosphatide emulsion hadfailed to clear appreciably from the liver, i.e., greater than 65% ofthe injected dose remained in the liver at this time point. On the otherhand, as shown on Table 4E, only 35% of the injected d6se delivered bythe TO/DOPC emulsion remained in the liver.

This distinction in kinetic biodistribution profile may be explained bythe fact that the majority of the iodinated triglyceride delivered inthe soybean oil/egg phosphatide emulsion was delivered to the RES cellsrather than the hepatocytes. If the radioactive material was deliveredto the Kupffer cells, following first pass clearance the imaging andbiodistribution profiles would, at early timepoints show excellent liverlocalization. However, with time, the material would not be cleared fromthe liver nearly as quickly as if it had been delivered to thehepatocytes. A comparison of Table 4C with Table 4F demonstrates thatthe TO/DOPC emulsion cleared from the liver quicker than the soybeanoil/egg phosphatide emulsion.

In addition, Folch lipid extraction analysis of liver and plasma samplesprovided information about the form of the iodinatedtriglyceride-derived radioactivity in each respective sample.

                  TABLE 5                                                         ______________________________________                                        Time After                                                                            Mean        Mean         Mean                                         Injection                                                                             Aqueous Conc.                                                                             Organic Conc.                                                                              ppt. Conc.                                   (Hours) (% of Total)                                                                              (% of Total) (% of Total)                                 ______________________________________                                        Liver                                                                         0.5     0.72% ± 0.11%                                                                          85.04% ± 1.49%                                                                          13.35% ± 1.56%                            3.0      1.8% ± 0.21%                                                                          88.52% ± 0.42%                                                                          10.40% ± 0.22%                            Plasma                                                                        0.5     24.96% ± 2.24%                                                                         57.88% ± 2.21%                                                                          17.16% ± 1.61%                            3.0     15.37% ± 1.91%                                                                         54.94% ± 1.55%                                                                          19.69% ± 1.46%                            ______________________________________                                    

The data in Table 5 confirm that there is a difference in the manner inwhich the two formulations were metabolized. At both 30 minutes and 3hours post-injection, an average of 1% of all liver-associatedradioactivity was water soluble, 12% was protein-associated, and 87% wasorganic soluble. Thus, the vast majority of liver-associatedtriglyceride was most likely the parent triglyceride. Results for theTO/DOPC emulsion were similar. However, in the plasma, there was noindication that metabolism of the iodinated triglyceride delivered withthe soy oil/egg phosphatide formulation occurred over time, in otherwords, the majority of radioactivity in the plasma (62% of the totaldose) was still organic soluble both 30 minutes and 3 hours afteradministration. On the other hand, the ITG delivered in the TO/DOPCemulsion exhibited a shift over time from being localized in the organicsoluble fraction to being localized in the protein-associated andwater-soluble fractions.

In addition to the foregoing, biliary and urinary elimination wasstudied by direct cannulation of the bile duct and ureters in rats towhich the radiolabeled emulsion of Example 2 was administered (27.8 mgI/kg body weight). This technique enabled bile and urine to be collecteddirectly and continuously for up to 3 hours after administration of thetest compound. The relative contributions of the biliary and urinarypathways to overall elimination of the compounds were measured. Theresults are reported in FIG. 19 which is a graphic representation of theelimination profile of the composition of Example 2 wherein cumulativeelimination (% dose) via the biliary route (▪) and the urinary route(▾), and bile flow in ml/min (), are shown as a function of time (mins)following injection the radioiodinated oil-in-water emulsion. Themajority of the iodinated triglyceride was eliminated by the biliaryroute, thus providing further evidence that the iodinated triglyceridehad been delivered primarily to the hepatocytes.

Fluorine Embodiments:

Non-proton magnetic resonance imaging of the liver has been accomplishedin the prior art with emulsified perfluorocarbons, such as Fluosol andperfluoroctylbromide, which are sequestered not only by hepatic Kupffercells but also by reticuloendothelial cells of the spleen and bonemarrow. In illustrative embodiments of the present invention,symmetrically substituted polyfluorinated triglycerides have beenincorporated into chylomicron-like microemulsions as useful as magneticresonance contrast agents.

EXAMPLE 11

In a specific illustrative embodiment, an oil-in-water emulsion containsa fluorinated triglyceride (FTG) which isglyceryl-2-oleoyl-1,3-bis(trifluoromethyl)phenyl acetate. Theoil-in-water emulsion contains 10% total core lipids, 2.4% DOPC, and hasa cholesterol to DOPC ratio of 0.4.

    ______________________________________                                                     Quantity                                                                             Percent (w/v)                                             ______________________________________                                        FTG            670.9 mg 6.7%                                                  Triolein       330.3 mg 3.3%                                                  Cholesterol     47.2 mg 0.5%                                                  α-Tocopherol                                                                            61.0 mg 0.6%                                                  DOPC           240.0 mg 2.4%                                                  Glycerol       500.7 mg 5.0%                                                  Sterile Water   8.6 ml                                                        ______________________________________                                    

The core lipids were combined and dissolved in ethyl acetate:ethanol(2:1, v/v) and then placed on a rotary evaporator to remove the solventmixture under vacuum. After addition of glycerol to the lipid mixture,emulsification was performed in the manner described in Example 2 toafford the finished oil-in-water emulsion.

EXAMPLE 12

In another specific example, fluorinated compounds of the type shown inU.S. Pat. Nos. 5,116,599 and 5,234,680 can be incorporated in anoil-in-water emulsion of the present invention. Specifically, the lipidcore components may include a fluorine-containing lipid, such as anester or triglyceride of a perfluoro-t-butyl-containing fatty acidcompounds, such as 7,7,7-trifluoro-6,6-bis (trifluoromethyl)-heptanoitacid or 8,8,8-tri fluoro-7,7-bis(trifluoromethyl)-octanoic acid.

The oil-in-water emulsions of the present invention are suitable forparentdral administration to a mammalian subject, typically byintravenous administration. However, intramuscular, subcutaneous,intraperitoneal, and other delivery routes are within the contemplationof the invention. Further, the oil-in-water emulsions of the presentinvention may be administered by other routes, such as oral.

Anticipated dose levels are 20 to 150 mgI/kg body weight. In theradioiodinated embodiments reported herein, specific activity for theradioactive forms ranges from about 16 to 20 μCi/ml.

In a method of use aspect of the invention, delivery of agents to theintracellular space of specific cell types within tissues could provideprobes for assessment of the metabolic and/or biochemical activity ofthe tissue. An example of such an application of the invention is theintracellular delivery of metabolically active diagnostic imagingagents. Comparisons between clearance rates of an intracellular agentand image enhancement in normal and disease conditions could provideinformation about the physiological state of the tissue. If a compoundthat requires, for example, enzymatic hydrolysis for clearance from thetissue were selected as the agent to be delivered to the tissue, thecompound would be cleared less effectively under specific diseaseconditions that decrease the levels or activities of the specificmetabolic enzymes. Suppressed enzyme concentrations and/or activitiesare associated with many hepatic diseases so that the intracellulardelivery of compounds in accordance with the present invention couldprovide a powerful means of assessing metabolic function in livertissue.

It is further contemplated that additional target specificity may begained by adding apolipoproteins or peptides incorporating the relevantrecognition and targeting portions of the apolipoproteins into theamphipathic monolayer of the particle. The addition of steryl esters tothe core of the particle, for example, might also influence thetargeting of the vehicle to specific tissues by mimicking otherlipoprotein classes, including, but not limited to, LDL or HDL.

While the invention has been presented in terms of ahepatocyte-selective delivery vehicle, it is possible to apply theprinciples of the invention to the production of tissue-specificdelivery vehicles for other tissues, such as the spleen, adrenals,prostate, ovaries, lymph nodes, etc. Tissue-specific targeting compoundsexploit existing cellular uptake pathways which are unique to eachtissue type in an analogous manner to the exploitation of the lipidmetabolism of liver tissue using the chylomicron remnant receptors onhepatocytes.

Further it should be noted that the animal models selected and used inthe studies presented hereinabove, specifically rats and dogs, are wellknown to have hepatic physiologies that closely resemble the hepaticphysiology of humans.

Although the invention has been described in terms of specificembodiments and applications, persons skilled in the art can, in lightof this teaching, generate additional embodiments without exceeding thescope or departing from the spirit of the claimed invention.Accordingly, it is to be understood that the drawing and description inthis disclosure are proffered to facilitate comprehension of theinvention, and should not be construed to limit the scope thereof.

What is claimed is:
 1. A method of making a heat stablehepatocyte-selective oil-in-water emulsion comprising the steps of:(a)preblending the lipophilic components of an oil-in-water emulsionincluding nonpolar core lipids, polar lipid emulsifiers, and otherlipophilic components to form a preblended lipid phase; (b) homogenizingthe preblended lipid phase and sodium ion-free aqueous components toform a crude oil-in-water emulsion; and (c) subjecting the crudeoil-in-water emulsion to ultra high energy mixing emulsification toproduce a fine oil-in-water emulsion having a mean particle diameter ofthe oil phase between 50 to 200 nm with greater than 98% of theparticles being less that 300 nm.
 2. The method of claim 1 comprisingthe further step of:subjecting the fine oil-in-water emulsion tosequential filtering.
 3. The method of claim 1 wherein the step ofpreblending comprises the steps of:mixing the lipophilic components withone or more organic solvents in an amount sufficient to facilitateblending of the lipophilic components; and removing the organicsolvents.
 4. The method of claim 1 wherein the step of homogenizingcomprises:adding an aqueous osmolality adjusting agent and mixing thelipophilic components and osmolality adjusting agent in a high speedmixer at a first speed; and adding the remainder of the aqueouscomponents and mixing in the high speed mixer at a second speed inexcess of the first speed.
 5. The method of claim 4 wherein the highspeed mixer is operated at a first speed of about 12.500 rpm for atleast about 5 minutes at a temperature which is greater than or equal tothe transition temperature or melting point of the lipophilic componentsand less than the temperature at which any one of the lipophiliccomponents will degrade.
 6. The method of claim 5 wherein the secondspeed is about 25,000 rpm for at least about 5 minutes.
 7. The method ofclaim 4 wherein the high speed mixer is a Polytron homogenizer.
 8. Themethod of claim 1 wherein the step of subjecting comprises:processingthe crude oil-in-water emulsion in an ultra high pressure homogenizer.9. The method of claim 8 wherein the ultra high pressure homogenizer isoperated in a recycling mode at a pressure of between about 10,000 and30,000 psi and temperature between about 30° C. and 60° C. for up to 20minutes.
 10. The method of claim 9 wherein the pressure is between about12,500 and 18,200 psi.
 11. The method of claim 8 wherein the ultra highpressure homogenizer is a Microfluidizer.
 12. The method of claim 1further including the step of:sterilizing the fine oil-in-wateremulsion.
 13. The method of claim 12 wherein said step of sterilizingcomprises heat sterilization.
 14. A heat stable hepatocyte-selectiveoil-in-water emulsion made by a method a comprising the steps of:a)preblending the lipophilic components of the oil-in-water emulsion, thelipophilic components comprising triolein and a polyhalogenatedtriglyceride in a molar ratio of 1:1, the triolein and polyhalogenatedtriglyceride comprising an amount of up to 50% (w/v) of the totalemulsion with a phospholipid emulsifier, the phospholipid being in anamount of up to 10% (w/v) and cholesterol, so that cholesterol comprisesless than about 5% (w/v) and is present in a molar ratio of cholesterolto phospholipid of 0.4; b) adding the aqueous components to thepreblended lipophilic components, the aqueous components being up to 5%(w/v) glycerol and the remainder water; c) homogenizing the preblendedlipid phase and aqueous components to form a crude oil-in-wateremulsion; (d) subjecting the crude oil-in-water emulsion to ultra highenergy mixing to produce a fine oil-in-water emulsion having a meanparticle diameter of the oil phase between 50 to 200 nm with greaterthan 98% of the particles being less that 300 nm; and (e) filtering thefine oil-in-water emulsion through sequentially through filters havingsmaller pores.